Therapeutic agents targeting the NCCa-ATP channel and methods of use thereof

ABSTRACT

The present invention is directed to therapeutic compositions targeting the NC Ca-ATP  channel of an astrocyte, neuron or capillary endothelial cell and methods of using same. More specifically, antagonists of the NC Ca-ATP  channel are contemplated. The compositions are used to prevent cell death and to treat secondary damage associated with spinal cord injury.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application claims priority to U.S. Provisional Application No.60/610,758 filed Sep. 18, 2004 and 60/698,272 filed Jul. 11, 2005, eachof which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made in part with government support under Grant No.NS048260 awarded by the National Institutes of Health and a Merit Reviewgrant from the United States Department of Veterans Affairs. The UnitedStates Government may have certain rights in the invention.

STATEMENT REGARDING OTHER SPONSORED RESEARCH OR DEVELOPMENT

This invention was made in part with support under a grant from theChristopher Reeves Paralysis Foundation (CRPF). The CRPF may havecertain rights in the invention.

TECHNICAL FIELD

The present invention is directed to fields of cell biology, physiologyand medicine. More specifically, the present invention addresses novelmethods of treating a patient comprising administering a therapeuticcompound that targets a unique non-selective cation calcium-ATP channel(NC_(Ca-ATP) channel) found in astrocytes. In specific embodiments, thetherapeutic compound is an antagonist, and uses thereof in therapies,such as treatment of spinal cord injury, benefiting from blocking and/orinhibiting the NC_(Ca-ATP) channel. Compositions comprising of theNC_(Ca-ATP) channel are also contemplated.

BACKGROUND OF THE INVENTION

NC_(Ca-ATP) Channel

A unique non-selective monovalent cationic ATP senstive channel(NC_(Ca-ATP) channel) was identified first in native reactive astrocytes(NRAs) and later, as described herein, in neurons and capillaryendothelial cells after stroke or traumatic brain injury (See,International application WO 03/079987 to Simard et al., and Chen andSimard, 2001, each incorporated by reference herein in its entirety).The NC_(Ca-ATP) channel is thought to be a heteromultimer structurecomprised of sulfonylurea receptor type 1 (SUR1) regulatory subunits andpore-forming subunits, similar to the K_(ATP) channel in pancreatic βcells (Chen et al., 2003). The pore-forming subunits of the NC_(Ca-ATP)channel remain uncharacterized.

SUR imparts sensitivity to antidiabetic sulfonylureas such asglibenclamide and tolbutamide, and is responsible for activation by achemically diverse group of agents termed “K⁺ channel openers” such asdiazoxide, pinacidil and cromakalin (Aguilar-Bryan et al., 1995; Inagakiet al., 1996; Isomoto et al., 1996; Nichols et al., 1996; Shyng et al.,1997). In various tissues, molecularly distinct SURs are coupled todistinct pore-forming subunits to form different K_(ATP) channels withdistinguishable physiological and pharmacological characteristics. TheK_(ATP) channel in pancreatic β cells is formed from SUR1 linked withKir6.2, whereas the cardiac and smooth muscle K_(ATP) channels areformed from SUR2A and SUR2B linked with Kir6.2 and Kir6.1, respectively(Fujita et al., 2000). Despite being made up of distinctly differentpore-forming subunits, the NC_(Ca-ATP) channel is also sensitive tosulfonylurea compounds.

Also, unlike the K_(ATP) channel, the NC_(Ca-ATP) channel conductssodium ions, potassium ions, cesium ions and other monovalent cationswith near equal facility (Chen and Simard, 2001) suggesting further thatthe characterization, and consequently the affinity to certaincompounds, of the NC_(Ca-ATP) channel differs from the K_(ATP) channel.

Other nonselective cation channels that are activated by intracellularCa²⁺ and inhibited by intracellular ATP have been identified but not inastrocytes. Further, the NC_(Ca-ATP) channel expressed and found inastrocytes differs physiologically from the other channels with respectto calcium sensitivity and adenine nucleotide sensitivity (Chen et al.,2001).

Other nonselective cation channels that are activated by intracellularCa²⁺ and inhibited by intracellular ATP have been identified inendothelial cells (Csanady and Adam-Vizi, Biophysical Journal,85:313-327, 2003), but these channels are not regulated by SUR1 and arenot inhibited by glibenclamide.

Spinal Cord Injury

A contusion injury to the spinal cord is often worsened by secondarydamage from tissue inflammation and swelling. Secondary injury thatexpands the region of irreversible damage should, in principal, bepreventable since it occurs in delayed fashion while under medical care,but effective treatments are not yet available. Secondary injurytypically involves a zone of potentially viable tissue, called thepenumbra, that surrounds the initial injury. Viability of neural tissuesin the penumbra is precarious, and those tissues can easily succumb anddie.

Changes in gene expression related to inflammation are among theearliest and strongest responses following spinal cord injury (Bareyreand Schwab, 2003; Bartholdi and Schwab, 1997).

An inflammatory response is necessary for resolution of the pathogenicevent, but bystander or collateral tissue damage is caused by the toxicnature of many of its by-products. It is generally recognized thatinflammation can be deleterious because cytotoxic agents such as TNFαand NO may be released, and because inflammation promotes formation ofedema and swelling, which in turn contribute to tissue ischemia. Thus, astrong inflammatory response can cause expansion of the original zone oftissue death. In contrast, ameliorating the inflammatory response candiminish the overall extent of damage.

One of the most potent stimulators of inflammation in spinal cord injuryis blood that extravasates from fractured capillaries following injury.Blood is universally held to be highly toxic to central nervous systemtissues, include spinal cord.

Cells die by apoptosis and necrosis. The distinction is important, notso much for cells that die, but for cells in surrounding tissues—thepenumbra—that may survive, albeit tenuously at first. Necrotic deathincites an inflammatory response, whereas apoptotic death does not.Molecular mechanisms responsible for inflammation following necroticcell death are not fully understood, but it is likely that necroticdeath, unlike apoptotic death, is accompanied by release ofintracellular molecules when cell membranes lyse. These intracellularmolecules, when released, activate other cells, notably microglia, whoseactivation results in expression of chemokines that in turn attractinflammatory cells. Thus, a logical therapeutic goal is to reducenecrosis, even if only to convert it to apoptosis, to reduce the releaseof intracellular molecules that initiate inflammation.

An important class of intracellular molecules that can initiateinflammation in necrotic death is heat shock proteins (HSP). Injury tothe spinal cord causes activation of astrocytes and up-regulation ofdevelopmentally regulated intracellular proteins, including vimentin,nestin and HSP. HSP-32 and HSP-70 are of special interest because theyare up-regulated in spinal cord injury (Song et al., 2001; Mautes etal., 2000; Mautes and Noble, 2000). In astrocytes, HSP-32 (hemeoxygenase-1) is induced by blood and blood products, and HSP-70 isinduced by hypoxia or glucose deprivation (Regan et al., 2000; Matz etal., 1996; Lee et al., 2001; Currie et al., 2000; Xu and Giffard, 1997;Papadopoulos et al., 1996; Copin et al., 1995).

HSP-70 and HSP-32 activate microglia in vivo, (Kakimura et al., 2002)and activated microglia, in turn, release inflammatory chemokines thatattract macrophages and polymorphonuclear leukocytes (PMNs). Thus,deleterious pathological events leading to inflammation-mediatedsecondary injury may originate, in part, with necrotic death ofastrocytes and release of HSPs as well as from extravasated blood.Therefore, the present invention is directed to decreasing necroticdeath of reactive astrocytes and to reducing extravasation of blood asan improved therapeutic strategy to treat spinal cord injury.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to therapeutic compositions comprisingan antagonist of a NC_(Ca-ATP) channel of neuronal cell, a neurogliacell or an endothelial cell.

The present invention is directed to methods of reducing spinal cordinjury in a patient in need thereof comprising administering anantagonist of a NC_(Ca-ATP) channel of a neuronal cell, neuroglia cellor an endothelial cell. The antagonist inhibits (closes, blocks,deactivates, decreases biological activity) the NC_(Ca-ATP) channel. Thespinal cord injury may comprises a contusion on the spinal cord.

One embodiment of the present invention comprises a method of treating asubject suffering from a spinal cord injury comprising administering tothe subject a compound effective to inhibit a NC_(Ca-ATP) channel in aneuronal cell, a neuroglia cell, a neural endothelial cell or acombination thereof. The compound effectively inhibits the NC_(Ca-ATP)channel by closing, blocking, partially blocking, and/or deactivatingthe channel thereby decreasing the Na+ influx, as well as othermonovalent ion influx, into the cell, decreasing the accumulation ofwater in the cell thereby decreasing cell swelling. Thus, the compoundof the present invention reduces, decreases or inhibits the activationof the NC_(Ca-ATP) channel which reduces an influx of sodium ions (Na⁺)thereby reducing and/or preventing or lessening the depolarization ofthe cell.

The subject can comprise a subject suffering from a spinal injury or asubject at risk for a spinal injury. Subjects at risk can include thosesubjects that are undergoing a surgical treatment and/or a radiationtreatment. Other subjects at risk can include subjects having a spinalcondition, for example, segmental deformities, cord compressions causedby any known type of disease or infection. For example, Cushing'ssyndrome can result in a growth of epidural fat tissue that compressesthe spinal cord. Other diseases could include arthritic diseases of thespine.

The composition of the present invention may be delivered alimentary orparenterally. Examples of alimentary administration include, but are notlimited to orally, buccally, rectally, or sublingually. Parenteraladministration can include, but are not limited to intramuscularly,subcutaneously, intraperitoneally, intravenously, intratumorally,intraarterially, intraventricularly, intracavity, intravesical,intrathecal, or intrapleural. Other modes of administration may alsoinclude topically, mucosally (i.e., intranasally), or transdermally.

An effective amount of an antagonist of NC_(Ca-ATP) channel that may beadministered to a cell includes a dose of about 0.0001 nM to about 2000μM. More specifically, doses to be administered are from about 0.01 nMto about 2000 μM; about 0.01 μM to about 0.05 μM; about 0.05 μM to about1.0 μM; about 1.0 μM to about 1.5 μM; about 1.5 μM to about 2.0 μM;about 2.0 μM to about 3.0 μM; about 3.0 μM to about 4.0 μM; about 4.0 μMto about 5.0 μM; about 5.0 μM to about 10 μM; about 10 μM to about 50μM; about 50 μM to about 100 μM; about 100 μM to about 200 μM; about 200μM to about 300 μM; about 300 μM to about 500 μM; about 500 μM to about1000 μM; about 1000 μM to about 1500 μM and about 1500 μM to about 2000μM. Of course, all of these amounts are exemplary, and any amountin-between these points is also expected to be of use in the invention.

An effective amount of an antagonist of the NC_(Ca-ATP) channel orrelated-compounds thereof as a treatment varies depending upon the hosttreated and the particular mode of administration. In one embodiment ofthe invention the dose range of the antagonist of the NC_(Ca-ATP)channel or related-compounds thereof will be about 0.01 μg/kg bodyweight to about 20,000 μg/kg body weight. The term “body weight” isapplicable when an animal is being treated. When isolated cells arebeing treated, “body weight” as used herein should read to mean “totalcell body weight”. The term “total body weight” may be used to apply toboth isolated cell and animal treatment. All concentrations andtreatment levels are expressed as “body weight” or simply “kg” in thisapplication are also considered to cover the analogous “total cell bodyweight” and “total body weight” concentrations. However, those of skillwill recognize the utility of a variety of dosage range, for example,0.01 μg/kg body weight to 20,000 μg/kg body weight, 0.02 μg/kg bodyweight to 15,000 μg/kg body weight, 0.03 μg/kg body weight to 10,000μg/kg body weight, 0.04 μg/kg body weight to 5,000 μg/kg body weight,0.05 μg/kg body weight to 2,500 μg/kg body weight, 0.06 μg/kg bodyweight to 1,000 μg/kg body weight, 0.07 μg/kg body weight to 500 μg/kgbody weight, 0.08 μg/kg body weight to 400 μg/kg body weight, 0.09 μg/kgbody weight to 200 μg/kg body weight or 0.1 μg/kg body weight to 100μg/kg body weight. Further, those of skill will recognize that a varietyof different dosage levels will be of use, for example, 0.0001 μg/kg,0.0002 μg/kg, 0.0003 μg/kg, 0.0004 μg/kg, 0.005 μg/kg, 0.0007 μg/kg,0.001 μg/kg, 0.1 μg/kg, 1.0 μg/kg, 1.5 μg/kg, 2.0 μg/kg, 5.0 μg/kg, 10.0μg/kg, 15.0 μg/kg, 30.0 μg/kg, 50 μg/kg, 75 μg/kg, 80 μg/kg, 90 μg/kg,100 μg/kg, 120 μg/kg, 140 μg/kg, 150 μg/kg, 160 μg/kg, 180 μg/kg, 200μg/kg, 225 μg/kg, 250 μg/kg, 275 μg/kg, 300 μg/kg, 325 μg/kg, 350 μg/kg,375 μg/kg, 400 μg/kg, 450 μg/kg, 500 μg/kg, 550 μg/kg, 600 μg/kg, 700μg/kg, 750 μg/kg, 800 μg/kg, 900 μg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 12mg/kg, 15 mg/kg, 20 mg/kg, and/or 30 mg/kg. Of course, all of thesedosages are exemplary, and any dosage in-between these points is alsoexpected to be of use in the invention. Any of the above dosage rangesor dosage levels may be employed for an antagonist of NC_(Ca-ATP)channel or related-compounds thereof.

The NC_(Ca-ATP) channel is blocked or deactivated or inhibited byantagonists of type 1 sulfonylurea receptor (SUR1) and opened by SUR1activators. More specifically, the antagonists of type 1 sulfonylureareceptor (SUR1) include blockers of K_(ATP) channels and the SUR1activators include activators of K_(ATP) channels. More specifically,the NC_(Ca-ATP) channel of the present invention has a single-channelconductance to potassium ion (K⁺) between 20 and 50 pS. The NC_(Ca-ATP)channel is also stimulated by Ca²⁺ on the cytoplasmic side of the cellmembrane in a physiological concentration range, where concentrationrange is from 10⁻⁸ to 10⁻⁵ M. The NC_(Ca-ATP) channel is also inhibitedby cytoplasmic ATP in a physiological concentration range, where theconcentration range is from 10⁻¹ to 10 M. The NC_(Ca-ATP) channel isalso permeable to the following cations; K⁺, Cs⁺, Li⁺, Na⁺; to theextent that the permeability ratio between any two of the cations isgreater than 0.5 and less than 2.

The channel can be inhibited (closed, deactivated, blocked, partiallyinhibited or blocked, etc.) by an NC_(Ca-ATP) channel inhibitor, anNC_(Ca-ATP) channel blocker, a type 1 sulfonylurea receptor (SUR1)antagonist, SUR1 inhibitor, or a compound capable of reducing themagnitude of membrane current through the channel. More specifically,the SUR1 antagonist is selected from the group consisting ofglibenclamide, tolbutamide, repaglinide, nateglinide, meglitinide,midaglizole, LY397364, LY389382, glyclazide, glimepiride, estrogen,estrogen related-compounds (estradiol, estrone, estriol, genistein,non-steroidal estrogen (e.g., diethystilbestrol), phytoestrogen (e.g.,coumestrol), zearalenone, etc.), and compounds known to inhibit or blockK_(ATP) channels. MgADP can also be used to inhibit the channel. Othercompounds that can be used to block or inhibit K_(ATP) channels include,but are not limited to tolbutamide, glyburide(1[p-2[5-chloro-O-anisamido)ethyl] phenyl]sulfonyl]-3-cyclohexyl-3-urea); chlopropamide(1-[[(p-chlorophenyl)sulfonyl]-3-propylurea; glipizide(1-cyclohexyl-3[[p-[2(5-methylpyrazine carboxamido)ethyl] phenyl]sulfonyl] urea); ortolazamide(benzenesulfonamide-N-[[(hexahydro-1H-azepin-1yl)amino]carbonyl]-4-methyl).

In certain embodiments, the amount of the SUR1 antagonist administeredto the subject is in the range of about 0.0001 μg/kg/day to about 20mg/kg/day, about 0.01 μg/kg/day to about 100 μg/kg/day, or about 100μg/kg/day to about 20 mg/kg/day. Still further, the SUR1 antagonist maybe administered to the subject in the from of a treatment in which thetreatment may comprise the amount of the SUR1 antagonist or the dose ofthe SUR1 antagonist that is administered per day (1, 2, 3, 4, etc.),week (1, 2, 3, 4, 5, etc.), month (1, 2, 3, 4, 5, etc.), etc. Treatmentsmay be administered such that the amount of SUR1 antagonist administeredto the subject is in the range of about 0.0001 μg/kg/treatment to about20 mg/kg/treatment, about 0.01 μg/kg/treatment to about 100μg/kg/treatment, or about 100 μg/kg/treatment to about 20mg/kg/treatment.

In certain embodiments, the antagonist treats adverse conditionsassociated with cytotoxic and ionic edema of the central nervous system.Such conditions include trauma, spinal cord injury, namely secondaryneuronal injury, for example, but not limited to hemorrhagic conversion,immune system reactions, oxidative damage, calcium and excitotoxicity,necrosis and apoptosis, and/or axon damage. The protection viadeactivation and/or inhibition of the NC_(Ca-ATP) channel is associatedwith a reduction in edema, reduction in cell death, reduction inextrvasation of blood in the injury site, reduction in the generation ofreactive oxidative species, reduction in inflammation or theinflammatory response, and/or reduction in hemorrhagic conversion. Thus,the compound of the present invention reduces these symptoms compared tothe level of the symptoms if the compound was not administered.

In certain embodiments, the NC_(Ca-ATP) channel is blocked, inhibited,or otherwise is decreased in activity such that the ion Na⁺ and/or othermonovalent ions influx through the channel is reduced, ceased, decreasedand/or stopped. The antagonist may prevent or lessen the depolarizationof the cells thereby lessening cell swelling due to osmotic changes thatcan result from Na⁺ influx and depolarization of the cells. Thus,inhibition of the NC_(CA-ATP) channel can reduce cytotoxic edema anddeath of cells, for example, necrotic death of cells. Thus, theantagonist of the present invention can be used to reduce secondarydamage associated with the spinal cord injury.

Still further, the present invention may comprise methods to reduce ordecrease the morbidity of a subject suffering from a spinal cord injurycomprising administering an effective amount of a compound to inhibitand/or deactivate the NC_(Ca-ATP) channel in a neuronal cell, aneuroglia cell, an endothelial cell or a combination thereof. Areduction in morbidity results in a improvement in physical and/ormovement outcomes and/or sensation of the subject. Thus, an increase inthe movement range and/or an increase in the sensation of the subject isan indicator that morbidity is reduced. In further embodiments, anincrease in the physical well-being of the subject is also an indicatorthat the morbidity of the subject is reduced.

Still further, the present invention can comprise a method of reducingthe blood and/or hemoglobin concentration in or near or surrounding thecontusion site of a subject suffering a spinal cord injury comprisingadministering an effective amount of a compound to inhibit theNC_(Ca-ATP) channel in a neuronal cell, a neuroglia cell, an endothelialcell or a combination thereof.

Yet further, another embodiment comprises a method of reducing thelesion size of a spinal cord injury in a subject comprisingadministering an effective amount of a compound to inhibit theNC_(Ca-ATP) channel in a neuronal cell, a neuroglia cell, an endothelialcell or a combination thereof. A reduction in the lesion size reducesthe likelihood of contralateral involvement.

Another embodiment of the present invention comprises increase orimproving the preservation of myelinated long tracts comprisingadministering an effective amount of a compound to inhibit theNC_(Ca-ATP) channel in a neuronal cell, a neuroglia cell, an endothelialcell or a combination thereof.

Still further, another embodiment of the present invention comprises themethod of decreasing the up-regulation of GFAP in a subject sufferingfrom a spinal cord injury comprising administering an effective amountof a compound to inhibit the NC_(Ca-ATP) channel in a neuronal cell, aneuroglia cell, an endothelial cell or a combination thereof.

Yet further, another embodiment comprises a method of reducingextravasation of blood from a spinal cord injury comprisingadministering an effective amount of a compound to inhibit theNC_(Ca-ATP) channel in a neuronal cell, a neuroglia cell, an endothelialcell or a combination thereof. The subject may be a subject that issuffering from a spinal cord injury or may be at risk for a spinal cordinjury, for example a subject undergoing surgery or radiation. Thus, thecompound may be administered before, during or after a surgical and/orradiation treatment.

Another embodiment of the present invention comprises a method ofreducing edema in the penumbra of the spinal cord injury in a subjectcomprising administering to the subject a compound effective to inhibita NC_(Ca-ATP) channel in a neuronal cell, a neuroglia cell, a neuralendothelial cell or a combination thereof.

Yet further, another embodiment of the present invention comprisestreating a subject at risk for a spinal cord injury comprisingadministering an effective amount of a compound to inhibit theNC_(Ca-ATP) channel in a neuronal cell, a neuroglia cell, an endothelialcell or a combination thereof. Subjects at risk can include thosesubjects that are undergoing a surgical treatment and/or a radiationtreatment. Other subjects at risk can include subjects having a spinalcondition, for example, segmental deformities, cord compressions causedby any known type of disease or infection, for example, Cushing'ssyndrome or arthritic diseases of the spine.

Still further, another embodiment of the present invention comprises amethod of diagnosing neuronal cell edema and/or cytotoxic damage in thespinal cord of a subject comprising: labeling an antagonist of SUR1;administering the labeled antagonist of SUR1 to the subject; measuringthe levels of labeled antagonist of SUR1 in the spinal cord of thesubject, wherein the presence of labeled antagonist of SUR1 in thespinal cord of the subject indicates neuronal cell edema and/orcytotoxic damage in the spinal cord. Labeled antagonist can include acompound labeled with a fluorescent marker and/or a radioactive marker.The compound may comprise an inhibitor of SUR1, an antibody of SUR1,and/or a nucleic acid molecule, etc.

Another embodiment comprises a method of determining the penumbrafollowing spinal cord injury in a subject comprising: labeling anantagonist of SUR1; administering the labeled antagonist of SUR1 to thesubject; visualizing the labeled antagonist of SUR1 in the spinal cordof the subject, wherein the presence of labeled antagonist of SUR1indicates the penumbra following a spinal cord injury in the subject.

In certain embodiments, determining the penumbra indicates the positionof neuronal damage and/or monitors disease progression.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawing, in which:

FIGS. 1A-1C show scanning electron micrographs showing appearance offreshly isolated reactive astrocyte (FIG. 1A) and blebbing 5 min (FIG.1B) and 25 min (FIG. 1C) after exposure to 1 mM Na azide. Separatelabeling showed that cells were GFAP-positive.

FIG. 2 shows a phase contrast micrographs showing appearance of freshlyisolated reactive astrocytes under control conditions, and blebbingafter exposure to 1 mM Na azide. Blebbing was reproduced by diazoxidealone, which opens the NC_(Ca-ATP) channel, whereas Na-azide inducedblebbing was blocked by glibenclamide, which blocks the channel.Separate labeling showed that cells were GFAP-positive.

FIG. 3 shows that addition of exogenousphosphatidylinositol-4,5-bisphosphate (PIP₂) causes activation of theNC_(Ca-ATP) channel, despite the presence of ATP in the bath solution.Initially, channel activity was recorded in an inside-out patch ofmembrane from an R1 astrocyte, with a bath solution containing 1 μM Ca²⁺and 10 μM ATP, which was sufficient to block channel activity. Additionof 50 μM PIP₂ resulted in channel activation, reflecting an apparentdecrease in affinity of the channel for ATP.

FIG. 4 shows that the NC_(Ca-ATP) channel in an R1 astrocyte isinhibited by estrogen. The initial portion of the record shows briskactivity from a number of superimposed channels, recorded in a cellattached patch of membrane from an R1 astrocyte obtained from a female.Addition of 10 nM estrogen to the bath promptly resulted in stronginhibition of channel activity. The mechanism involved is believed to berelated to estrogen receptor mediated activation of phospholipase C(PLC), resulting in depletion of PIP₂ from the membrane, and reflectingan apparent increase in affinity for ATP.

FIGS. 5A-5B show Western blots demonstrating that R1 astrocytes fromboth males and females express estrogen receptors and SUR1, a marker ofthe NC_(Ca-ATP) channel. Cell lysates were obtained from gelatin spongeimplants from males (M) and females (F) and studied at two dilutions (4×and 1×), with lysates from uterus used as controls. FIG. 5A wasdeveloped using antibodies directed against estrogen receptors (ER),demonstrating that both ERα and ERβ are expressed in astrocytes fromboth genders. Western blots showed that SUR1 is also expressed by cellsfrom both genders, with pancreatic tissue used as control (FIG. 5B).

FIG. 6 shows that the NC_(Ca-ATP) channel in an R1 astrocyte from a maleis inhibited by estrogen. The initial portion of the record shows briskactivity from a number of superimposed channels, recorded in a cellattached patch of membrane from an R1 astrocyte obtained from a male.Addition of 10 nM estrogen to the bath promptly resulted in stronginhibition of channel activity.

FIGS. 7A-7B shows Na azide-induced blebbing is followed by necroticdeath of freshly isolated reactive astrocytes. Cell death was assessedusing propidium iodide (PI) to identify necrotic death (FIG. 7A) andannexin V to identify apoptotic death (FIG. 7B). The significant rise innecrotic death induced by 1 mM Na azide was strongly attenuated by 1 μMglibenclamide (FIG. 7A). Apoptotic death was minimal after exposure toNa azide (FIG. 7B).

FIGS. 8A-8B shows immunofluorescence images of 1 cell in the penumbra(FIG. 8A) and 2 cells in the middle of a stroke in the brain (FIG. 8B,8C), immunolabeled for SUR1; co-labeling with GFAP confirmed theiridentity as astrocytes; note “bleb-like” pattern of labeling

FIGS. 9A-9B show images of traumatic brain injury site after infusion ofdiazoxide to induce necrotic death of reactive astrocytes. Sections werelabeled with the nuclear marker, DAPI, showing sheets of small cells(FIG. 9A), and immunolabeled with anti-MMP-8 antibody, to identify thecells as PMNs (FIG. 9B).

FIGS. 10A-10D show immunofluorescence (composite) images of spinal cordsections from control (FIG. 10A) and 24-hr after severe bilateralthoracic spinal cord crush injury (FIGS. 10B-10D), labeled for SUR1(FIGS. 10A, 10B, 10D) or GFAP (FIG. 10C). At high magnification,individual SUR1-positive loci seen in (FIG. 10B) correspond toGFAP-positive stellate cells (FIG. 10D) consistent with reactiveastrocytes.

FIGS. 11A-11C shows SUR1 up-regulation in a cervical hemi-spinal cordcontusion injury of moderate severity (SCI; same model as used in allsubsequent illustrations). Epifluorescence images of spinal cord tissuesimmunolabeled for SUR1 from control region (FIG. 11A) and from region ofcontusion injury, shown at low power (FIG. 11B) and at high power (FIG.11C). The high power view demonstrates that in this model, SUR1expression at 24 hr occurs primarily in capillaries.

FIGS. 12A-12H show SUR1 and vimentin are up-regulated in capillaries inSCI. Epifluorescence images of spinal cord tissues from 2 ratsimmunolabeled for SUR1 (FIGS. 12A, 12D) and coimmunolabeled for vimentin(FIGS. 12B, 12E, 12F) 24 hr after contusion injury; superimposed imagesare also shown.

FIGS. 13A-B show up-regulation of the transcription factor, SP1, whichis the principal transcription factor known to regulate expression ofSUR1. Epifluorescence images of spinal cord tissues from 2 ratsimmunolabeled for SP1, a control uninjured spinal cord (FIG. 13A) and aspinal cord 24 hr after contusion injury (FIG. 13B).

FIGS. 14A-14C show glibenclamide treatment reduces hemorrhagicconversion. FIGS. 14A and 14B show images of frozen sections of spinalcord 24 hr after contusion injury for a rat treated with saline (FIG.14A) and a rat treated with glibenclamide (FIG. 14B); note the smallerhemorrhage and preservation of contralateral structures withglibenclamide treatment. FIG. 14C shows test tubes containinghomogenates of spinal cord 24 hr after contusion injury, for 2 ratstreated with saline (left) and 2 rats treated with glibenclamide(right); note the difference in color, reflecting an apparent reductionin hemoglobin concentration with glibenclamide treatment.

FIG. 15 shows the effect of glibenclamide treatment on the time courseof hemorrhagic conversion after spinal cord injury (SCI). Extravasatedblood in the region of injury was assessed at various times after SCI.At the time of sacrifice, intravascular blood was first removed byperfusion, then a 5 mm segment of spinal cord encompassing the contusedarea was excised, weighed and homogenized in a volume of distilled water9× the mass of the tissue. The content of blood was quantified usingDrabkin reagent (5 rats per group). Values were expressed as absorbanceat 560 nm or as microliters of blood, assuming a hematocrit of 40%. Insaline-treated animals, the amount of blood increased progressively withtime following SCI, reaching a plateau 6 hours after injury (filledsquares). In glibenclamide-treated animals, values at 45 min weresimilar to controls, but as time progressed after SCI, values increasedsignificantly less than in controls (filled circles).

FIGS. 16A-16D show glibenclamide treatment reduces lesion size, GFAPexpression, and preserves contralateral long tracks. FIGS. 16A and 16Bshow epifluorescence images of spinal cord sections 24 hr aftercontusion injury, immunolabeled for glial fibrillary acidic protein(GFAP), in a rat treated with saline (FIG. 16A) and a rat treated withglibenclamide (FIG. 16B). FIGS. 16C and 16D show images of spinal cordsections 24 hr after contusion injury, stained for myelin usingeriochrome cyanine-R, in a rat treated with saline (FIG. 16C) and a rattreated with glibenclamide (FIG. 16D). Note the smaller lesions andsparing of contralateral structures with glibenclamide.

FIG. 17 shows glibenclamide treatment improves vertical explorationfollowing SCI contusion. Bar graphs showing the number of seconds spentin vertical exploration (rearing) per 3-min period of observation, 24 hrafter contusion injury, for 6 rats treated with saline, and for 5 ratstreated with glibenclamide.

DETAILED DESCRIPTION OF THE INVENTION I. DEFINITIONS

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. For purposes of the presentinvention, the following terms are defined below.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternative are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.”

As used herein, the term “antagonist” refers to a biological or chemicalagent that acts within the body to reduce the physiological activity ofanother chemical or biological substance. The term antagonist includes,but is not limited small molecules, chemicals, proteins, peptides,nucleic acid molecules, etc. In the present invention, the antagonistblocks, inhibits, reduces and/or decreases the activity of a NC_(Ca-ATP)channel of a neuronal cell, a neuroglia cell or a neural endothelialcell (e.g., capillary endothelial cells). In the present invention, theantagonist combines, binds, associates with a NC_(Ca-ATP) channel ofneuronal cell, a neuroglia cell or a neural endothelial cell (e.g.,capillary endothelial cells), such that the NC_(Ca-ATP) channel isclosed (deactivated, partially blocked, blocked, or inhibited), meaningreduced biological activity with respect to the biological activity inthe diseased state. In certain embodiments, the antagonist combines,binds and/or associates with a regulatory subunit of the NC_(Ca-ATP)channel, particularly a SUR1. Alternatively, the antagonist combines,binds, and/or associates with a pore-forming subunit of the NC_(Ca-ATP)channel, such that the NC_(Ca-ATP) channel is closed (deactivated and/orinhibited). The terms antagonist or inhibitor can be usedinterchangeably.

As used herein, the term “depolarization” refers to an increase in thepermeability of the cell membrane to sodium ions wherein the electricalpotential difference across the cell membrane is reduced or eliminated.

As used herein, the terms “effective amount” or “therapeuticallyeffective amount” are interchangeable and refer to an amount thatresults in an improvement or remediation of the symptoms of the diseaseor condition. Those of skill in the art understand that the effectiveamount may improve the patient's or subject's condition, but may not bea complete cure of the disease and/or condition.

As used herein, the term “endothelium” refers a layer of cells that linethe inside surfaces of body cavities, blood vessels, and lymph vesselsor that form capillaries.

As used herein, the term “endothelial cell” refers to a cell of theendothelium or a cell that lines the surfaces of body cavities, forexample, blood or lymph vessels or capillaries. In certain embodiments,the term endothelial cell refers to a neural endothelial cell or anendothelial cell that is part of the nervous system, for example thecentral nervous system, i.e., the spinal cord.

As used herein, the term “hemorrhagic conversion” refers to thepathological sequence that takes place in capillaries after ischemia.One of skill in the art is aware that hemorrhagic conversion is due tocatastrophic failure of capillaries, during which all constituents ofblood extravasate into the surrounding tissues. In accordance withStarling's law, understanding these phases requires that 2 things beidentified: (i) the driving force that “pushes” things into tissue; and(ii) the permeability pore that allows passage of these things intotissue.

As used herein, the term “inhibit” refers to the ability of the compoundto block, partially block, interfere, decrease, reduce or deactivate theNC_(Ca-ATP) channel. Thus, one of skill in the art understands that theterm inhibit encompasses a complete and/or partial loss of activity ofthe NC_(Ca-ATP) channel as indicated by the reduction in celldepolarization, reduction in sodium ion influx or any other monovalention influx, reduction in an influx of water, reduction in extravasationof blood, reduction in cell death, as well as an improvement.

As used herein, the term “lesion” refers to any pathological ortraumatic discontinuity of tissue or loss of function of a part thereof.For example, lesions includes any injury associated with the spinalcord, for example, but not limited to contusions, compression injuries,etc.

The term “morbidity” as used herein is the state of being diseased. Yetfurther, morbidity can also refer to the disease rate or the ratio ofsick subjects or cases of disease in to a given population.

The term “mortality” as used herein is the state of being mortal orcausing death. Yet further, mortality can also refer to the death rateor the ratio of number of deaths to a given population.

As used herein, the term “neuronal cell” refers to a cell that is amorphologic and functional unit of the nervous system. The cellcomprises a nerve cell body, the dendrites, and the axon. The termsneuron, nerve cell, neuronal, neurone, and neurocyte can be usedinterchangeably. Neuronal cell types can include, but are not limited toa typical nerve cell body showing internal structure, a horizontal cell(of Cajal) from cerebral cortex; Martinottic cell, bipolar cell,unipolar cell, Pukinje cell, and a pyramidal cell of motor area ofcerebral cortex.

As used herein, the term “neural” refers to anything associated with thenervous system.

As used herein, the terms “neuroglia” or “neuroglial cell” refers to acell that is a non-neuronal cellular element of the nervous system. Theterms neuroglia, neurogliacyte, and neuroglial cell can be usedinterchangeably. Neuroglial cells can include, but are not limited toependymal cells, astrocytes, oligodendrocytes, or microglia.

As used herein, the term “reduces” refers to a decrease in cell death,inflammatory response, hemorrhagic conversion, extravasation of blood,etc as compared to no treatment with the compound of the presentinvention. Thus, one of skill in the art is able to determine the scopeof the reduction of any of the symptoms and/or conditions associatedwith a spinal cord injury in which the subject has received thetreatment of the present invention compared to no treatment and/or whatwould otherwise have occurred without intervention.

The term “preventing” as used herein refers to minimizing, reducing orsuppressing the risk of developing a disease state or parametersrelating to the disease state or progression or other abnormal ordeleterious conditions.

As used herein, “spinal cord,” “spinal nervous tissue associated with avertebral segment,” “nervous tissue associated with a vertebral segment”or “spinal cord associated with a vertebral segment or level” includesany spinal nervous tissue associated with a vertebral level or segment,all of which are interchangeable. Thus, one of skill in the art is awarethat spinal tissue includes all the neuronal cells, as well as any ofthe neuroglia cells associated therewith. Those of skill in the art areaware that the spinal cord and tissue associated therewith areassociated with cervical, thoracic and lumbar vertebrae. As used herein,C1 refers to cervical vertebral segment 1, C2 refers to cervicalvertebral segment 2, and so on. T1 refers to thoracic vertebral segment1, T2 refers to thoracic vertebral segment 2, and so on. L1 refers tolumbar vertebral segment 1, L2 refers to lumbar vertebral segment 2, andso on, unless otherwise specifically noted.

The term “subject” as used herein, is taken to mean any mammaliansubject to which the composition is administered according to themethods described herein. A skilled artisan realizes that a mammaliansubject, includes, but is not limited to humans, monkeys, horses, pigs,cows, dogs, cats, rats and mice. In a specific embodiment, the methodsof the present invention are employed to treat a human subject. Infurther embodiments, the subject is at risk of developing a spinal cordinjury. Thus, the subject may or may not be cognizant of their diseasestate or potential disease state and may or may not be aware that theyare need of treatment (therapeutic treatment or prophylactic treatment).

The terms “treating” and “treatment” as used herein refer toadministering to a subject a therapeutically effective amount of acomposition so that the subject has an improvement in the disease orcondition. The improvement is any observable or measurable improvement.Thus, one of skill in the art realizes that a treatment may improve thepatient's condition, but may not be a complete cure of the disease.Treating may also comprise treating subjects at risk of developing adisease and/or condition.

II. THE PRESENT INVENTION

The present invention is directed to therapeutic compositions andmethods of using the same. In one embodiment, the therapeuticcomposition is an antagonist of a NC_(Ca-ATP) channel of a neuronalcell, a neuroglial cell, or a neural endothelial cell.

In certain embodiments, the therapeutic compound of the presentinvention comprises an antagonist of a NC_(Ca-ATP) channel of a neuronalcell, a neuroglia cell or an endothelial cell. Antagonists arecontemplated for use in treating adverse conditions associated withcytotoxic and ionic edema of the central nervous system. Such conditionsinclude trauma, spinal cord injury, namely secondary neuronal injury,for example, but not limited to hemorrhagic conversion, immune systemreactions, oxidative damage, calcium and excitotoxicity, necrosis andapoptosis, and/or axon damage. The protection via inhibition of theNC_(Ca-ATP) channel is associated with a reduction in edema, reductionin the generation of reactive oxidative species, reduction in incitinginflammation, and/or reduction in hemorrhagic conversion.

In one aspect, the NC_(Ca-ATP) channel is blocked, inhibited, orotherwise is decreased in activity. In such examples, an antagonist ofthe NC_(Ca-ATP) channel is administered and/or applied. The antagonistmodulates the NC_(Ca-ATP) channel such that flux through the channel isreduced, ceased, decreased and/or stopped. The antagonist may have areversible or an irreversible activity with respect to the activity ofthe NC_(Ca-ATP) channel of the neuronal cell, neuroglial cell,endothelial cell or a combination thereof. The antagonist may prevent orlessen the depolarization of the cells thereby lessening cell swellingdue to osmotic changes that can result from Na⁺ influx anddepolarization of the cells. Thus, inhibition of the NC_(Ca-ATP) channelcan reduce cytotoxic edema and death of cells, for example, necroticdeath of cells.

In a preferred embodiment, the present invention provides a method ofreducing spinal cord injury in a patient comprising administering anantagonist of a NC_(Ca-ATP) channel of a neuronal cell, a neurogliacell, or an endothelial cell, wherein the antagonist binds the channel.The binding of the NC_(Ca-ATP) channel blocks the influx of Na⁺ andwater into the astrocyte, neuronal cell and endothelial cell, therebyreducing swelling at or around the injury. More particularly, theantagonist reduces the secondary injury from the initial spinal cordinjury, for example, reduces the progression of a pathologicalinvolvement of the capillaries, i.e., hemorrhagic conversion, reducesimmune system reactions, reduces oxidative damage, reduces calcium andexcitotoxicity, reduces necrosis and cell death, and/or reduces axondamage.

III. NC_(Ca-ATP) CHANNEL

The invention is based, in part, on the discovery of a specific channel,the NC_(Ca-ATP) channel, defined as a channel on astrocytes in USApplication Publication No. 20030215889, which is incorporated herein byreference in its entirety. More specifically, the present invention hasfurther defined that this channel is not only expressed on astrocytes,it is expressed on neural cells, neuroglial cells, and/or neuralendothelial cells after central nervous system trauma, for example, aspinal cord contusion, or other secondary neuronal injuries relating tothese events.

The NC_(Ca-ATP) channel is activated by calcium ions (Ca²⁺) and issensitive to ATP. Thus, this channel is a non-selective cation channelactivated by intracellular Ca²⁺ and blocked by intracellular ATP. Whenopened by depletion of intracellular ATP, this channel is responsiblefor complete depolarization due to massive Na⁺ influx, which creates anelectrical gradient for Cl⁻ and an osmotic gradient for H₂O, resultingin cytotoxic edema and cell death. When the channel is blocked orinhibited, massive Na⁺ does not occur thereby preventing cytotoxicedema.

Certain functional characteristics distinguishes the NC_(Ca-ATP) channelfrom other known ion channels. These characteristics can include, butare not limited to 1) it is a non-selective cation channels that readilyallows passage of Na⁺, K⁺ and other monovalent cations; 2) it isactivated by a decrease in intracellular ATP in the presence ofintracellular Ca²⁺; 3) it is regulated by sulfonylurea receptor type 1(SUR1), which heretofore had been considered to be associatedexclusively with K_(ATP) channels such as those found in pancreatic βcells.

More specifically, the NC_(Ca-ATP) channel of the present invention hasa single-channel conductance to potassium ion (K⁺) between 20 and 50 pS.The NC_(Ca-ATP) channel is also stimulated by Ca²⁺ on the cytoplasmicside of the cell membrane in a physiological concentration range, whereconcentration range is from 10⁻⁸ to 10⁻⁵ M. The NC_(Ca-ATP) channel isalso inhibited by cytoplasmic ATP in a physiological concentrationrange, where the concentration range is from 10⁻¹ to 10 M. TheNC_(Ca-ATP) channel is also permeable to the following cations; K⁺, Cs⁺,Li⁺, Na⁺; to the extent that the permeability ratio between any two ofthe cations is greater than 0.5 and less than 2.

IV. INHIBITORS OF THE NC_(Ca-ATP) CHANNEL

The present invention comprises inhibitors of the channel, for examplean antagonist of the channel. Examples of antagonists of the presentinvention may encompass antagonists identified in US ApplicationPublication No. 20030215889, which is incorporated herein by referencein its entirety. One of skill in the art is aware that the NC_(Ca-ATP)channel is comprised to two subunits, the regulatory subunit, SUR1, andthe pore forming subunit. One of skill in the art is aware that thenucleic acid sequences and amino acid sequences for SUR1 are readilyavailable in GenBank, for example, GenBank accession L40624 (GI:1311533)and AAA99237 (GI:1311534), each of which is incorporated herein byreference in its entirety.

A. Inhibitors of SUR1

In certain embodiments, antagonists to sulfonylurea receptor-1 (SUR1)are suitable for blocking the channel. Examples of suitable SUR1antagonists include, but are not limited to glibenclamide, tolbutamide,repaglinide, nateglinide, meglitinide, midaglizole, LY397364, LY389382,glyclazide, glimepiride, estrogen, estrogen related-compounds(estradiol, estrone, estriol, genistein, non-steroidal estrogen (e.g.,diethystilbestrol), phytoestrogen (e.g., coumestrol), zearalenone, etc.)and combinations thereof. In a preferred embodiment of the invention theSUR1 antagonists is selected from the group consisting of glibenclamideand tolbutamide. Yet further, another antagonist can be MgADP. Otherantagonist include blockers of K_(ATP) channels, for example, but notlimited to tolbutamide, glyburide (1[p-2[5-chloro-O-anisamido)ethyl]phenyl] sulfonyl]-3-cyclohexyl-3-urea); chlopropamide(1-[[(p-chlorophenyl)sulfonyl]-3-propylurea; glipizide (1-cyclohexyl-3[[p-[2(5-methylpyrazine carboxamido) ethyl] phenyl] sulfonyl] urea); ortolazamide(benzenesulfonamide-N-[[(hexahydro-1H-azepin-1yl)amino]carbonyl]-4-methyl).

B. Inhibitors of SUR1 Transcription and/or Translation

In certain embodiments, the inhibitor can be a compound (protein,nucleic acid, siRNA, etc.) that modulates transcription and/ortranslation of SUR1 (regulatory subunit) and/or the molecular entitiesthat comprise the pore-forming subunit.

1. Transcription Factors

Transcription factors are regulatory proteins that binds to a specificDNA sequence (e.g., promoters and enhancers) and regulate transcriptionof an encoding DNA region. Thus, transcription factors can be used tomodulate the expression of SUR1. Typically, a transcription factorcomprises a binding domain that binds to DNA (a DNA binding domain) anda regulatory domain that controls transcription. Where a regulatorydomain activates transcription, that regulatory domain is designated anactivation domain. Where that regulatory domain inhibits transcription,that regulatory domain is designated a repression domain.

More specifically, transcription factors such as Sp1 and HIF1α can beused to modulate expression of SUR1. Those of skill in the art recognizethat Sp1 and HIF1α can regulate SUR1 expression. Thus, one could preventexpression/activation of Sp1 that is normally induced byischemia/hypoxia and/or hyperglycemia, that would in turn preventexpression of SUR1 (Chae Y M et al., 2004, incorporated herein byreference). Thus, inhibitors or molecules that prevent binding of Sp1and/or HIF1 are contemplated in the present invention. Other suchinhibitors of Sp1 can include, but are not limited to mithramycin.

Thus, it is contemplated that a candidate substance or SUR1 inhibitormay be a DNA-binding protein or transcription factor or a molecule orcompound that inhibits or interferes with the activity or binding of atranscription factor, such as Sp1 or HIF1. It is proposed that the SUR1inhibitor may bind to regulatory elements located within genes to altertranscription of the gene or may prevent the binding of DNA-bindingproteins or transcription factors, such as Sp1 or HIF1. Alsocontemplated in the present invention is the interaction of a putativeSUR1 inhibitor with another compound, e.g., a protein, to form acomplex, which interacts with the DNA to alter transcription, such asprevents or reduces transcription. It will be understood that thecompound that interacts with the putative SUR1 inhibitor may one or morethan one compound.

2. Antisense and Ribozymes

An antisense molecule that binds to a translational or transcriptionalstart site, or splice junctions, are ideal inhibitors. Antisense,ribozyme, and double-stranded RNA molecules target a particular sequenceto achieve a reduction or elimination of a particular polypeptide, suchas SUR1. Thus, it is contemplated that antisense, ribozyme, anddouble-stranded RNA, and RNA interference molecules are constructed andused to modulate SUR1.

a. Antisense Molecules

Antisense methodology takes advantage of the fact that nucleic acidstend to pair with complementary sequences. By complementary, it is meantthat polynucleotides are those which are capable of base-pairingaccording to the standard Watson-Crick complementarity rules. That is,the larger purines will base pair with the smaller pyrimidines to formcombinations of guanine paired with cytosine (G:C) and adenine pairedwith either thymine (A:T) in the case of DNA, or adenine paired withuracil (A:U) in the case of RNA. Inclusion of less common bases such asinosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others inhybridizing sequences does not interfere with pairing.

Targeting double-stranded (ds) DNA with polynucleotides leads totriple-helix formation; targeting RNA will lead to double-helixformation. Antisense polynucleotides, when introduced into a targetcell, specifically bind to their target polynucleotide and interferewith transcription, RNA processing, transport, translation and/orstability. Antisense RNA constructs, or DNA encoding such antisenseRNAs, are employed to inhibit gene transcription or translation or bothwithin a host cell, either in vitro or in vivo, such as within a hostanimal, including a human subject.

Antisense constructs are designed to bind to the promoter and othercontrol regions, exons, introns or even exon-intron boundaries of agene. It is contemplated that the most effective antisense constructsmay include regions complementary to intron/exon splice junctions. Thus,antisense constructs with complementarity to regions within 50-200 basesof an intron-exon splice junction are used. It has been observed thatsome exon sequences can be included in the construct without seriouslyaffecting the target selectivity thereof. The amount of exonic materialincluded will vary depending on the particular exon and intron sequencesused. One can readily test whether too much exon DNA is included simplyby testing the constructs in vitro to determine whether normal cellularfunction is affected or whether the expression of related genes havingcomplementary sequences is affected.

It is advantageous to combine portions of genomic DNA with cDNA orsynthetic sequences to generate specific constructs. For example, wherean intron is desired in the ultimate construct, a genomic clone willneed to be used. The cDNA or a synthesized polynucleotide may providemore convenient restriction sites for the remaining portion of theconstruct and, therefore, would be used for the rest of the sequence.

b. RNA Interference

It is also contemplated in the present invention that double-strandedRNA is used as an interference molecule, e.g., RNA interference (RNAi).RNA interference is used to “knock down” or inhibit a particular gene ofinterest by simply injecting, bathing or feeding to the organism ofinterest the double-stranded RNA molecule. This technique selectively“knock downs” gene function without requiring transfection orrecombinant techniques (Giet, 2001; Hammond, 2001; Stein P, et al.,2002; Svoboda P, et al., 2001; Svoboda P, et al., 2000).

Another type of RNAi is often referred to as small interfering RNA(siRNA), which may also be utilized to inhibit SUR1. A siRNA maycomprises a double stranded structure or a single stranded structure,the sequence of which is “substantially identical” to at least a portionof the target gene (See WO 04/046320, which is incorporated herein byreference in its entirety). “Identity,” as known in the art, is therelationship between two or more polynucleotide (or polypeptide)sequences, as determined by comparing the sequences. In the art,identity also means the degree of sequence relatedness betweenpolynucleotide sequences, as determined by the match of the order ofnucleotides between such sequences. Identity can be readily calculated.See, for example: Computational Molecular Biology, Lesk, A. M., ed.Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ea., Academic Press, New York, 1993, andthe methods disclosed in WO 99/32619, WO 01/68836, WO 00/44914, and WO01/36646, specifically incorporated herein by reference. While a numberof methods exist for measuring identity between two nucleotidesequences, the term is well known in the art. Methods for determiningidentity are typically designed to produce the greatest degree ofmatching of nucleotide sequence and are also typically embodied incomputer programs. Such programs are readily available to those in therelevant art. For example, the GCG program package (Devereux et al.),BLASTP, BLASTN, and FASTA (Atschul et al.,) and CLUSTAL (Higgins et al.,1992; Thompson, et al., 1994).

Thus, siRNA contains a nucleotide sequence that is essentially identicalto at least a portion of the target gene, for example, SUR1, or anyother molecular entity associated with the NC_(Ca-ATP) channel such asthe pore-forming subunit. One of skill in the art is aware that thenucleic acid sequences for SUR1 are readily available in GenBank, forexample, GenBank accession L40624 (GI:1311533), which is incorporatedherein by reference in its entirety. Preferably, the siRNA contains anucleotide sequence that is completely identical to at least a portionof the target gene. Of course, when comparing an RNA sequence to a DNAsequence, an “identical” RNA sequence will contain ribonucleotides wherethe DNA sequence contains deoxyribonucleotides, and further that the RNAsequence will typically contain a uracil at positions where the DNAsequence contains thymidine.

One of skill in the art will appreciate that two polynucleotides ofdifferent lengths may be compared over the entire length of the longerfragment. Alternatively, small regions may be compared. Normallysequences of the same length are compared for a final estimation oftheir utility in the practice of the present invention. It is preferredthat there be 100% sequence identity between the dsRNA for use as siRNAand at least 15 contiguous nucleotides of the target gene (e.g., SUR1),although a dsRNA having 70%, 75%, 80%, 85%, 90%, or 95% or greater mayalso be used in the present invention. A siRNA that is essentiallyidentical to a least a portion of the target gene may also be a dsRNAwherein one of the two complementary strands (or, in the case of aself-complementary RNA, one of the two self-complementary portions) iseither identical to the sequence of that portion or the target gene orcontains one or more insertions, deletions or single point mutationsrelative to the nucleotide sequence of that portion of the target gene.siRNA technology thus has the property of being able to toleratesequence variations that might be expected to result from geneticmutation, strain polymorphism, or evolutionary divergence.

There are several methods for preparing siRNA, such as chemicalsynthesis, in vitro transcription, siRNA expression vectors, and PCRexpression cassettes. Irrespective of which method one uses, the firststep in designing an siRNA molecule is to choose the siRNA target site,which can be any site in the target gene. In certain embodiments, one ofskill in the art may manually select the target selecting region of thegene, which may be an ORF (open reading frame) as the target selectingregion and may preferably be 50-100 nucleotides downstream of the “ATG”start codon. However, there are several readily available programsavailable to assist with the design of siRNA molecules, for examplesiRNA Target Designer by Promega, siRNA Target Finder by GenScriptCorp., siRNA Retriever Program by Imgenex Corp., EMBOSS siRNA algorithm,siRNA program by Qiagen, Ambion siRNA predictor, Ambion siRNA predictor,Whitehead siRNA prediction, and Sfold. Thus, it is envisioned that anyof the above programs may be utilized to produce siRNA molecules thatcan be used in the present invention.

c. Ribozymes

Ribozymes are RNA-protein complexes that cleave nucleic acids in asite-specific fashion. Ribozymes have specific catalytic domains thatpossess endonuclease activity (Kim and Cech, 1987; Forster and Symons,1987). For example, a large number of ribozymes accelerate phosphoestertransfer reactions with a high degree of specificity, often cleavingonly one of several phosphoesters in an oligonucleotide substrate (Cechet al., 1981; Reinhold-Hurek and Shub, 1992). This specificity has beenattributed to the requirement that the substrate bind via specificbase-pairing interactions to the internal guide sequence (“IGS”) of theribozyme prior to chemical reaction.

Ribozyme catalysis has primarily been observed as part of sequencespecific cleavage/ligation reactions involving nucleic acids (Joyce,1989; Cech et al., 1981). For example, U.S. Pat. No. 5,354,855 reportsthat certain ribozymes can act as endonucleases with a sequencespecificity greater than that of known ribonucleases and approachingthat of the DNA restriction enzymes. Thus, sequence-specificribozyme-mediated inhibition of gene expression is particularly suitedto therapeutic applications (Scanlon et al., 1991; Sarver et al., 1990;Sioud et al., 1992). Most of this work involved the modification of atarget mRNA, based on a specific mutant codon that is cleaved by aspecific ribozyme. In light of the information included herein and theknowledge of one of ordinary skill in the art, the preparation and useof additional ribozymes that are specifically targeted to a given genewill now be straightforward.

Other suitable ribozymes include sequences from RNase P with RNAcleavage activity (Yuan et al., 1992; Yuan and Altman, 1994), hairpinribozyme structures (Berzal-Herranz et al., 1992; Chowrira et al., 1993)and hepatitis δ virus based ribozymes (Perrotta and Been, 1992). Thegeneral design and optimization of ribozyme directed RNA cleavageactivity has been discussed in detail (Haseloff and Gerlach, 1988;Symons, 1992; Chowrira, et al., 1994; and Thompson, et al., 1995).

The other variable on ribozyme design is the selection of a cleavagesite on a given target RNA. Ribozymes are targeted to a given sequenceby virtue of annealing to a site by complimentary base pairinteractions. Two stretches of homology are required for this targeting.These stretches of homologous sequences flank the catalytic ribozymestructure defined above. Each stretch of homologous sequence can vary inlength from 7 to 15 nucleotides. The only requirement for defining thehomologous sequences is that, on the target RNA, they are separated by aspecific sequence which is the cleavage site. For hammerhead ribozymes,the cleavage site is a dinucleotide sequence on the target RNA, uracil(U) followed by either an adenine, cytosine or uracil (A, C or U;Perriman, et al., 1992; Thompson, et al., 1995). The frequency of thisdinucleotide occurring in any given RNA is statistically 3 out of 16.

Designing and testing ribozymes for efficient cleavage of a target RNAis a process well known to those skilled in the art. Examples ofscientific methods for designing and testing ribozymes are described byChowrira et al. (1994) and Lieber and Strauss (1995), each incorporatedby reference. The identification of operative and preferred sequencesfor use in SUR1 targeted ribozymes is simply a matter of preparing andtesting a given sequence, and is a routinely practiced screening methodknown to those of skill in the art.

C. Methods of Screening for Inhibitors

Further embodiments of the present invention can include methods foridentifying inhibitors of the NC_(Ca-ATP) channel, for example,antagonists, that modify the activity and/or expression. These assaysmay comprise random screening of large libraries of candidatesubstances; alternatively, the assays may be used to focus on particularclasses of compounds selected with an eye towards structural attributesthat are believed to make them more likely to modulate the function oractivity or expression of the NC_(Ca-ATP) channel.

By function, it is meant that one may assay for mRNA expression, proteinexpression, protein activity, or channel activity, more specifically,the ability of the modulator to inhibit or block the NC_(Ca-ATP)channel. Thus, the compounds for screening in accordance with theinvention include, but are not limited to natural or synthetic organiccompounds, peptides, antibodies and fragments thereof, peptidomimetics,that bind to the NC_(Ca-ATP) channel and either block the channel (e.g.,antagonists).

With reference to screening of compounds that affect the NC_(Ca-ATP)channel, libraries of known compounds can be screened, including naturalproducts or synthetic chemicals, and biologically active materials,including proteins, for compounds which are inhibitors or activators.Preferably, such a compound is an NC_(Ca-ATP) antagonist, which includesan NC_(Ca-ATP) channel inhibitor, an NC_(Ca-ATP) channel blocker, a SUR1antagonist, SUR1 inhibitor, and/or a compound capable of reducing themagnitude of membrane current through the channel.

Compounds may include, but are not limited to, small organic orinorganic molecules, compounds available in compound libraries, peptidessuch as, for example, soluble peptides, including but not limited tomembers of random peptide libraries; (see, e.g., Lam, K. S. et al.,1991, Nature 354: 82-84; Houghten, R. et al., 1991, Nature 354: 84-86),and combinatorial chemistry-derived molecular library made of D- and/orL-configuration amino acids, phosphopeptides (including, but not limitedto, members of random or partially degenerate, directed phosphopeptidelibraries; see, e.g., Songyang, Z. et al., 1993, Cell 72: 767-778),antibodies (including, but not limited to, polyclonal, monoclonal,humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb,F(ab′)₂ and FAb expression library fragments, and epitope-bindingfragments thereof).

Other compounds which can be screened in accordance with the inventioninclude but are not limited to small organic molecules that are able tocross the blood-brain barrier, gain entry into an appropriate neuralcell and affect the expression of the NC_(Ca-ATP) channel gene or someother gene involved in the NC_(Ca-ATP) channel activity (e.g., byinteracting with the regulatory region or transcription factors involvedin gene expression); or such compounds that affects the activity of theNC_(Ca-ATP) channel or the activity of some other intracellular factorinvolved in the NC_(Ca-ATP) channel activity.

To identify, make, generate, provide, manufacture or obtain aninhibitor, one generally will determine the activity of the NC_(Ca-ATP)channel in the presence, absence, or both of the candidate substance,wherein an inhibitor or antagonist is defined as any substance thatdown-regulates, reduces, inhibits, blocks or decreases the NC_(Ca-ATP)channel expression or activity. For example, a method may generallycomprise:

-   -   providing a candidate substance suspected inhibiting the        NC_(Ca-ATP) channel expression or activity in vitro or in vivo;    -   assessing the ability of the candidate substance to inhibit the        NC_(CA-ATP) channel expression or activity in vitro or in vivo;    -   selecting an inhibitor; and    -   manufacturing the inhibitor.

In certain embodiments, an alternative assessing step can be assessingthe ability of the candidate substance to bind specifically to theNC_(Ca-ATP) channel in vitro or in vivo;

In further embodiments, the NC_(Ca-ATP) channel may be provided in acell or a cell free system and the NC_(Ca-ATP) channel may be contactedwith the candidate substance. Next, the inhibitor is selected byassessing the effect of the candidate substance on the NC_(Ca-ATP)channel activity or expression. Upon identification of the inhibitor,the method may further provide manufacturing of the inhibitor.

V. TREATMENT OF SPINAL CORD INJURY

In other embodiments, the therapeutic compound of the present inventioncomprises an antagonist of a NC_(CA-ATP) channel of a neuronal cell, aneuroglial cell, a neural endothelial cell or a combination thereof.Antagonists are contemplated for use in treating adverse conditionsassociated with a spinal cord injury. Such conditions include secondarydamage associated with spinal cord injury, for example, but not limitedto cell edema, cell death (e.g., necrotic cell death), inflammation,oxidative damage, axon damage, hemorrhagic conversion, etc. Antagonistsprotect the cells expressing the NC_(Ca-ATP) channel, which is desirablefor clinical treatment in which ionic or cytotoxic edema is formed, inwhich capillary integrity is lost. The protection via inhibition of theNC_(Ca-ATP) channel is associated with a reduction in ionic andcytotoxic edema. Thus, the compound that inhibits the NC_(Ca-ATP)channel is neuroprotective.

In one aspect, the NC_(Ca-ATP) channel is blocked, inhibited, orotherwise is decreased in activity. In such examples, an antagonist ofthe NC_(Ca-ATP) channel is administered and/or applied. The antagonistmodulates the NC_(CA-ATP) channel such that flux (ion and/or water)through the channel is reduced, ceased, decreased and/or stopped. Theantagonist may have a reversible or an irreversible activity withrespect to the activity of the NC_(CA-ATP) channel of the neuronal cell,neuroglial cell, a neural endothelial cell or a combination thereof.Thus, inhibition of the NC_(CA-ATP) channel can reduce cytotoxic edemaand death of endothelial cells which are associated with formation ofionic edema and with hemorrhagic conversion.

Accordingly, the present invention is useful in the treatment oralleviation of inflammation associated with spinal cord injury.According to a specific embodiment of the present invention theadministration of effective amounts of the active compound can block thechannel, which if remained open leads to neuronal cell swelling and celldeath, which lead to initiation of the inflammatory response. A varietyof antagonists to SUR1 are suitable for blocking the channel. Examplesof suitable SUR1 antagonists include, but are not limited toglibenclamide, tolbutamide, repaglinide, nateglinide, meglitinide,midaglizole, LY397364, LY389382, glyclazide, glimepiride, estrogen,estrogen related-compounds and combinations thereof. In a preferredembodiment of the invention the SUR1 antagonists is selected from thegroup consisting of glibenclamide and tolbutamide. Another antagonistthat can be used is MgADP. Still other therapeutic “strategies” forpreventing neural cell swelling and cell death can be adopted including,but not limited to methods that maintain the neural cell in a polarizedstate and methods that prevent strong depolarization.

In further embodiments, inhibitors or antagonist of the NC_(Ca-ATP)channel can be used to reduce or alleviate or abrogate hemorrhagicconversion and/or extravasated blood near or surrounding the injurysite. With the administration of an antagonist of the NC_(Ca-ATP)channel, endothelial cell depolarization is abrogated, slowed, reducedor inhibited due to the opening of the NC_(Ca-ATP) channel. Thus,abrogation of cell depolarization results in abrogation or inhibition ofNa⁺ influx, which prevents a change in osmotic gradient therebypreventing an influx of water into the endothelial cell and stoppingcell swelling, blebbing and cytotoxic edema. Thus, preventing orinhibiting or attenuating endothelial cell depolarization can prevent orreduce hemorrhagic conversion and/or extravasted blood near orsurrounding the injury site.

Thus, the use of the antagonist or related-compounds thereof can reducethe mortality and/or morbidity of a subject suffering from a spinal cordinjury and/or rescue the penumbra area or prevent damage in the penumbraarea which comprises areas of tissue that are at risk of becomingirreversibly damaged.

Neuronal cells in which the antagonist of the NC_(CA-ATP) channel may beadministered may include any cell that expresses SUR1, for example anyneuronal cell, neuroglial cell or a neural endothelia cell.

Subjects that may be treated with the antagonist or related-compoundthereof include those that are suffering from a spinal cord injury.Other subjects that may be treated with the antagonist of the presentinvention include those subjects that are at risk or predisposed todeveloping a spinal cord injury, such as a subject that is undergoingsurgery of the spinal cord or radiation treatments to the spinal cord.In such cases, the subject may be treated with the antagonist orrelated-compound of the present invention prior to the actual treatment.Pretreatment can include administration of the antagonist and/orrelated-compound months (1, 2, 3, etc.), weeks (1, 2, 3, etc.), days (1,2, 3, etc.), hours (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), or minutes(15, 30, 60, 90, etc.) prior to the actual treatment or surgery orradiation treatment. Treatment of the antagonist and/or related-compoundcan continue during the treatment and/or surgery and after the treatmentand/or surgery until the risk of developing a spinal cord injury in thesubject is decreased, lessened or alleviated. Still further, othersubjects at risk for a spinal cord injury can include those subjectsthat have segmental deformities and/or other spinal conditions orcompression diseases, for example arthritis or Cushing's disease.

An effective amount of an antagonist of the NC_(Ca-ATP) channel that maybe administered to a cell includes a dose of about 0.0001 nM to about2000 μM. More specifically, doses to be administered are from about 0.01nM to about 2000 μM; about 0.01 μM to about 0.05 μM; about 0.05 μM toabout 1.0 μM; about 1.0 μM to about 1.5 μM; about 1.5 μM to about 2.0μM; about 2.0 μM to about 3.0 μM; about 3.0 μM to about 4.0 μM; about4.0 μM to about 5.0 μM; about 5.0 μM to about 10 μM; about 10 μM toabout 50 μM; about 50 μM to about 100 μM; about 100 μM to about 200 μM;about 200 μM to about 300 μM; about 300 μM to about 500 μM; about 500 μMto about 1000 μM; about 1000 μM to about 1500 μM and about 1500 μM toabout 2000 μM. Of course, all of these amounts are exemplary, and anyamount in-between these points is also expected to be of use in theinvention.

The antagonist or related-compound thereof can be administeredparenterally or alimentary. Parenteral administrations include, but arenot limited to intravenously, intradermally, intramuscularly,intraarterially, intrathecally, subcutaneous, or intraperitoneally U.S.Pat. Nos. 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363(each specifically incorporated herein by reference in its entirety).Alimentary administrations include, but are not limited to orally,buccally, rectally, or sublingually.

The administration of the therapeutic compounds and/or the therapies ofthe present invention may include systemic, local and/or regionaladministrations, for example, topically (dermally, transdermally), viacatheters, implantable pumps, etc. Alternatively, other routes ofadministration are also contemplated such as, for example, arterialperfusion, intracavitary, intraperitoneal, intrapleural,intraventricular and/or intrathecal. The skilled artisan is aware ofdetermining the appropriate administration route using standard methodsand procedures. Other routes of administration are discussed elsewherein the specification and are incorporated herein by reference.

Treatment methods will involve treating an individual with an effectiveamount of a composition containing an antagonist of NC_(Ca-ATP) channelor related-compound thereof. An effective amount is described,generally, as that amount sufficient to detectably and repeatedly toameliorate, reduce, minimize or limit the extent of a disease or itssymptoms. More specifically, it is envisioned that the treatment withthe an antagonist of NC_(Ca-ATP) channel or related-compounds thereofwill inhibit cell depolarization, inhibit Na⁺ influx, inhibit an osmoticgradient change, inhibit water influx into the cell, inhibit cytotoxiccell edema, decrease inflammation, inhibit or reduce oxidative damage orgeneration of reactive oxidative species, inhibit hemorrhagicconversion, decrease morbidity, and decrease mortality of the subject.

The effective amount of an antagonist of NC_(Ca-ATP) channel orrelated-compounds thereof to be used are those amounts effective toproduce beneficial results, particularly with respect to spinal cordinjury treatment, in the recipient animal or patient. Such amounts maybe initially determined by reviewing the published literature, byconducting in vitro tests or by conducting metabolic studies in healthyexperimental animals. Before use in a clinical setting, it may bebeneficial to conduct confirmatory studies in an animal model,preferably a widely accepted animal model of the particular disease tobe treated. Preferred animal models for use in certain embodiments arerodent models, which are preferred because they are economical to useand, particularly, because the results gained are widely accepted aspredictive of clinical value.

As is well known in the art, a specific dose level of active compoundssuch as an antagonist of the NC_(Ca-ATP) channel or related-compoundsthereof for any particular patient depends upon a variety of factorsincluding the activity of the specific compound employed, the age, bodyweight, general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination, and the severity ofthe particular disease undergoing therapy. The person responsible foradministration will determine the appropriate dose for the individualsubject. Moreover, for human administration, preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biologics standards.

One of skill in the art realizes that the effective amount of theantagonist or related-compound thereof can be the amount that isrequired to achieve the desired result: reduction inflammation,reduction in cell death, reduction in hemorrhagic conversion, reductionin extravasated blood, reduction the lesion size, reduction in theup-regulation of cGFAP, etc. This amount also is an amount thatmaintains a reasonable level of blood glucose in the patient, forexample, the amount of the antagonist maintains a blood glucose level ofat least 60 mmol/l, more preferably, the blood glucose level is maintainin the range of about 60 mmol/l to about 150 mmol/l. Thus, the amountsprevents the subject from becoming hypoglycemic. If glucose levels arenot normal, then one of skill in the art would administer either insulinor glucose, depending upon if the patient is hypoglycemic orhyperglycemic.

Thus, in certain embodiments, the present invention comprisesco-administration of an antagonist of the NC_(Ca-ATP) channel withglucose or related carbohydrate to maintain appropriate levels of serumglucose. Appropriate levels of blood glucose are within the range ofabout 60 mmol/l to about 150 mmol/liter. Thus, glucose or a relatedcarbohydrate is administered in combination to maintain the serumglucose within this range.

An effective amount of an antagonist of the NC_(Ca-ATP) channel orrelated-compounds thereof as a treatment varies depending upon the hosttreated and the particular mode of administration. In one embodiment ofthe invention the dose range of the antagonist of the NC_(Ca-ATP)channel or related-compounds thereof will be about 0.01 μg/kg bodyweight to about 20,000 μg/kg body weight. The term “body weight” isapplicable when an animal is being treated. When isolated cells arebeing treated, “body weight” as used herein should read to mean “totalcell body weight”. The term “total body weight” may be used to apply toboth isolated cell and animal treatment. All concentrations andtreatment levels are expressed as “body weight” or simply “kg” in thisapplication are also considered to cover the analogous “total cell bodyweight” and “total body weight” concentrations. However, those of skillwill recognize the utility of a variety of dosage range, for example,0.01 μg/kg body weight to 20,000 μg/kg body weight, 0.02 μg/kg bodyweight to 15,000 μg/kg body weight, 0.03 μg/kg body weight to 10,000μg/kg body weight, 0.04 μg/kg body weight to 5,000 μg/kg body weight,0.05 μg/kg body weight to 2,500 μg/kg body weight, 0.06 μg/kg bodyweight to 1,000 μg/kg body weight, 0.07 μg/kg body weight to 500 μg/kgbody weight, 0.08 μg/kg body weight to 400 μg/kg body weight, 0.09 μg/kgbody weight to 200 μg/kg body weight or 0.1 μg/kg body weight to 100μg/kg body weight. Further, those of skill will recognize that a varietyof different dosage levels will be of use, for example, 0.0001 μg/kg,0.0002 μg/kg, 0.0003 μg/kg, 0.0004 μg/kg, 0.005 μg/kg, 0.0007 μg/kg,0.001 μg/kg, 0.1 μg/kg, 1.0 μg/kg, 1.5 μg/kg, 2.0 μg/kg, 5.0 μg/kg, 10.0μg/kg, 15.0 μg/kg, 30.0 μg/kg, 50 μg/kg, 75 μg/kg, 80 μg/kg, 90 μg/kg,100 μg/kg, 120 μg/kg, 140 μg/kg, 150 μg/kg, 160 μg/kg, 180 μg/kg, 200μg/kg, 225 μg/kg, 250 μg/kg, 275 μg/kg, 300 μg/kg, 325 μg/kg, 350 μg/kg,375 μg/kg, 400 μg/kg, 450 μg/kg, 500 μg/kg, 550 μg/kg, 600 μg/kg, 700μg/kg, 750 μg/kg, 800 μg/kg, 900 μg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 12mg/kg, 15 mg/kg, 20 mg/kg, and/or 30 mg/kg. Of course, all of thesedosages are exemplary, and any dosage in-between these points is alsoexpected to be of use in the invention. Any of the above dosage rangesor dosage levels may be employed for an antagonist of NC_(Ca-ATP)channel or related-compounds thereof.

Administration of the therapeutic antagonist of NC_(Ca-ATP) channelcomposition of the present invention to a patient or subject will followgeneral protocols for the administration of therapies taking intoaccount the toxicity, if any, of the antagonist of the NC_(Ca-ATP)channel. It is expected that the treatment cycles would be repeated asnecessary. It also is contemplated that various standard therapies, aswell as surgical intervention, may be applied in combination with thedescribed therapy.

The treatments may include various “unit doses.” Unit dose is defined ascontaining a predetermined quantity of the therapeutic composition (anantagonist of the NC_(Ca-ATP) channel or its related-compounds thereof)calculated to produce the desired responses in association with itsadministration, e.g., the appropriate route and treatment regimen. Thequantity to be administered, and the particular route and formulation,are within the skill of those in the clinical arts. Also of import isthe subject to be treated, in particular, the state of the subject andthe protection desired. A unit dose need not be administered as a singleinjection but may comprise continuous infusion over a set period oftime.

VI. COMBINATION TREATMENTS

In the context of the present invention, it is contemplated that anantagonist of the NC_(Ca-ATP) channel or related-compounds thereof maybe used in combination with an additional therapeutic agent to moreeffectively treat a spinal cord injury. In some embodiments, it iscontemplated that a conventional therapy or agent, including but notlimited to, a pharmacological therapeutic agent may be combined with theantagonist or related-compound of the present invention.

Pharmacological therapeutic agents and methods of administration,dosages, etc. are well known to those of skill in the art (see forexample, the “Physicians Desk Reference”, Goodman & Gilman's “ThePharmacological Basis of Therapeutics”, “Remington's PharmaceuticalSciences”, and “The Merck Index, Eleventh Edition”, incorporated hereinby reference in relevant parts), and may be combined with the inventionin light of the disclosures herein. Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject, and suchindividual determinations are within the skill of those of ordinaryskill in the art.

Non-limiting examples of a pharmacological therapeutic agents that maybe used in the present invention include an anti-inflammatory agent.Anti-inflammatory agents include, but are not limited to non-steroidalanti-inflammatory agents (e.g., naproxen, ibuprofen, celeocobix) andsteroidal anti-inflammatory agents (e.g., glucocorticoids,dexamethasone, methylprednisolone).

Other agents that can be used in combination with the antagonist of thepresent invention can include, but are not limited to antioxidants,calcium blockers, drugs that control excitotoxicity, and drugs thatenhance axon signaling, such as 4-aminopyridine.

Still further other agents that can be used in combination with theantagonist may also include agents designed to promote regeneration byusing trophic factors, and growth-inhibiting substances.

Yet further, non-pharmacological interventions may also be used incombination with the antagonist of the present invention, such astransplantation, peripheral nerve grafts, hypothermia (cooling).

When an additional therapeutic agent, as long as the dose of theadditional therapeutic agent does not exceed previously quoted toxicitylevels, the effective amounts of the additional therapeutic agent maysimply be defined as that amount effective to reduce edema or reducesecondary injury when administered to an animal in combination withNC_(Ca-ATP) channel or related-compounds thereof. This may be easilydetermined by monitoring the animal or patient and measuring thosephysical and biochemical parameters of health and disease that areindicative of the success of a given treatment. Such methods are routinein animal testing and clinical practice.

To inhibit hemorrhagic conversion, reduce oxidative stress, reduce celldeath, reduce cell swelling, etc., using the methods and compositions ofthe present invention, one would generally contact a cell withantagonist of NC_(Ca-ATP) channel or related-compounds thereof incombination with an additional therapeutic agent, such as, ananti-inflammatory agent, etc. These compositions would be provided in acombined amount effective to inhibit hemorrhagic conversion, cellswelling, cell death, edema, etc. This process may involve contactingthe cells with NC_(Ca-ATP) channel or related-compounds thereof incombination with an additional therapeutic agent or factor(s) at thesame time. This may be achieved by contacting the cell with a singlecomposition or pharmacological formulation that includes both agents, orby contacting the cell with two distinct compositions or formulations,at the same time, wherein one composition includes an antagonist of theNC_(Ca-ATP) channel or derivatives thereof and the other includes theadditional agent.

Alternatively, treatment with an antagonist of NC_(Ca-ATP) channel orrelated-compounds thereof may precede or follow the additional agenttreatment by intervals ranging from minutes to hours to weeks to months.In embodiments where the additional agent is applied separately to thecell, one would generally ensure that a significant period of time didnot expire between the time of each delivery, such that the agent wouldstill be able to exert an advantageously combined effect on the cell. Insuch instances, it is contemplated that one would contact the cell withboth modalities within about 1-24 hr of each other and, more preferably,within about 6-12 hr of each other.

VII. FORMULATIONS AND ROUTES FOR ADMINISTRATION OF COMPOUNDS

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more inhibitors of the NC_(Ca-ATP) channel(antagonist) or related-compounds or additional agent dissolved ordispersed in a pharmaceutically acceptable carrier. The phrases“pharmaceutical or pharmacologically acceptable” refers to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, such as, forexample, a human, as appropriate. The preparation of an pharmaceuticalcomposition that contains at least one modulators of NC_(Ca-ATP) channel(antagonist and/or agonist) or related-compounds or additional activeingredient will be known to those of skill in the art in light of thepresent disclosure, as exemplified by Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, incorporated herein byreference. Moreover, for animal (e.g., human) administration, it will beunderstood that preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the pharmaceuticalcompositions is contemplated.

The inhibitors of the NC_(Ca-ATP) channel (antagonist) orrelated-compounds may comprise different types of carriers depending onwhether it is to be administered in solid, liquid or aerosol form, andwhether it need to be sterile for such routes of administration asinjection. The present invention can be administered intravenously,intradermally, transdermally, intrathecally, intraventricularly,intraarterially, intraperitoneally, intranasally, intravaginally,intrarectally, topically, intramuscularly, subcutaneously, mucosally,orally, topically, locally, inhalation (e.g., aerosol inhalation),injection, infusion, continuous infusion, localized perfusion bathingtarget cells directly, via a catheter, via a lavage, in cremes, in lipidcompositions (e.g., liposomes), or by other method or any combination ofthe forgoing as would be known to one of ordinary skill in the art (see,for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack PrintingCompany, 1990, incorporated herein by reference).

The inhibitors of the NC_(Ca-ATP) channel (i.e., antagonist) orrelated-compounds may be formulated into a composition in a free base,neutral or salt form. Pharmaceutically acceptable salts, include theacid addition salts, e.g., those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acids suchas for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine. Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as formulated for parenteraladministrations such as injectable solutions, or aerosols for deliveryto the lungs, or formulated for alimentary administrations such as drugrelease capsules and the like.

Further in accordance with the present invention, the composition of thepresent invention suitable for administration is provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable and includes liquid, semi-solid, i.e.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent or carrier is detrimental to the recipient or to thetherapeutic effectiveness of a the composition contained therein, itsuse in administrable composition for use in practicing the methods ofthe present invention is appropriate. Examples of carriers or diluentsinclude fats, oils, water, saline solutions, lipids, liposomes, resins,binders, fillers and the like, or combinations thereof. The compositionmay also comprise various antioxidants to retard oxidation of one ormore component. Additionally, the prevention of the action ofmicroorganisms can be brought about by preservatives such as variousantibacterial and antifungal agents, including but not limited toparabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol,sorbic acid, thimerosal or combinations thereof.

In accordance with the present invention, the composition is combinedwith the carrier in any convenient and practical manner, i.e., bysolution, suspension, emulsification, admixture, encapsulation,absorption and the like. Such procedures are routine for those skilledin the art.

In a specific embodiment of the present invention, the composition iscombined or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, i.e.,denaturation in the stomach. Examples of stabilizers for use in an thecomposition include buffers, amino acids such as glycine and lysine,carbohydrates such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of apharmaceutical lipid vehicle compositions that include inhibitors ofNC_(Ca-ATP) channel (antagonist) or related-compounds, one or morelipids, and an aqueous solvent. As used herein, the term “lipid” will bedefined to include any of a broad range of substances that ischaracteristically insoluble in water and extractable with an organicsolvent. This broad class of compounds are well known to those of skillin the art, and as the term “lipid” is used herein, it is not limited toany particular structure. Examples include compounds which containlong-chain aliphatic hydrocarbons and their derivatives. A lipid may benaturally occurring or synthetic (i.e., designed or produced by man).However, a lipid is usually a biological substance. Biological lipidsare well known in the art, and include for example, neutral fats,phospholipids, phosphoglycerides, steroids, terpenes, lysolipids,glycosphingolipids, glycolipids, sulphatides, lipids with ether andester-linked fatty acids and polymerizable lipids, and combinationsthereof. Of course, compounds other than those specifically describedherein that are understood by one of skill in the art as lipids are alsoencompassed by the compositions and methods of the present invention.

One of ordinary skill in the art would be familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the inhibitors of NC_(Ca-ATP) channel (antagonist)or related-compounds may be dispersed in a solution containing a lipid,dissolved with a lipid, emulsified with a lipid, mixed with a lipid,combined with a lipid, covalently bonded to a lipid, contained as asuspension in a lipid, contained or complexed with a micelle orliposome, or otherwise associated with a lipid or lipid structure by anymeans known to those of ordinary skill in the art. The dispersion may ormay not result in the formation of liposomes.

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeutic and/orprophylatic interventions, idiopathy of the patient and on the route ofadministration. Depending upon the dosage and the route ofadministration, the number of administrations of a preferred dosageand/or an effective amount may vary according to the response of thesubject. The practitioner responsible for administration will, in anyevent, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition may beprepared is such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimensmay be desirable.

A. Alimentary Compositions and Formulations

In preferred embodiments of the present invention, the antagonist orrelated-compounds of the NC_(Ca-ATP) channel are formulated to beadministered via an alimentary route. Alimentary routes include allpossible routes of administration in which the composition is in directcontact with the alimentary tract. Specifically, the pharmaceuticalcompositions disclosed herein may be administered orally, buccally,rectally, or sublingually. As such, these compositions may be formulatedwith an inert diluent or with an assimilable edible carrier, or they maybe enclosed in hard- or soft-shell gelatin capsule, or they may becompressed into tablets, or they may be incorporated directly with thefood of the diet.

In certain embodiments, the active compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tables,troches, capsules, elixirs, suspensions, syrups, wafers, and the like(Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515;5,580,579 and 5,792,451, each specifically incorporated herein byreference in its entirety). The tablets, troches, pills, capsules andthe like may also contain the following: a binder, such as, for example,gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; anexcipient, such as, for example, dicalcium phosphate, mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate or combinations thereof; a disintegrating agent, such as, forexample, corn starch, potato starch, alginic acid or combinationsthereof; a lubricant, such as, for example, magnesium stearate; asweetening agent, such as, for example, sucrose, lactose, saccharin orcombinations thereof; a flavoring agent, such as, for examplepeppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.When the dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar, or both. When the dosage form is a capsule, it maycontain, in addition to materials of the above type, carriers such as aliquid carrier. Gelatin capsules, tablets, or pills may be entericallycoated. Enteric coatings prevent denaturation of the composition in thestomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No.5,629,001. Upon reaching the small intestines, the basic pH thereindissolves the coating and permits the composition to be released andabsorbed by specialized cells, e.g., epithelial enterocytes and Peyer'spatch M cells. A syrup of elixir may contain the active compound sucroseas a sweetening agent methyl and propylparabens as preservatives, a dyeand flavoring, such as cherry or orange flavor. Of course, any materialused in preparing any dosage unit form should be pharmaceutically pureand substantially non-toxic in the amounts employed. In addition, theactive compounds may be incorporated into sustained-release preparationand formulations.

For oral administration the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. For example, a mouthwash may beprepared incorporating the active ingredient in the required amount inan appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as one containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, or added in atherapeutically-effective amount to a composition that may includewater, binders, abrasives flavoring agents, foaming agents, andhumectants. Alternatively the compositions may be fashioned into atablet or solution form that may be placed under the tongue or otherwisedissolved in the mouth.

Additional formulations which are suitable for other modes of alimentaryadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum. After insertion, suppositories soften, melt or dissolvein the cavity fluids. In general, for suppositories, traditionalcarriers may include, for example, polyalkylene glycols, triglyceridesor combinations thereof. In certain embodiments, suppositories may beformed from mixtures containing, for example, the active ingredient inthe range of about 0.5% to about 10%, and preferably about 1% to about2%.

B. Parenteral Compositions and Formulations

In further embodiments, the antagonist or related-compounds ofNC_(Ca-ATP) channel may be administered via a parenteral route. As usedherein, the term “parenteral” includes routes that bypass the alimentarytract. Specifically, the pharmaceutical compositions disclosed hereinmay be administered for example, but not limited to intravenously,intradermally, intramuscularly, intraarterially, intraventricularly,intrathecally, subcutaneous, or intraperitoneally U.S. Pat. Nos.6,7537,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363(each specifically incorporated herein by reference in its entirety).

Solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy injectability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, DMSO, polyol (i.e., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. A powdered composition is combined with a liquidcarrier such as, e.g., water or a saline solution, with or without astabilizing agent.

C. Miscellaneous Pharmaceutical Compositions and Formulations

In other preferred embodiments of the invention, the antagonist orrelated-compounds of NC_(Ca-ATP) channel may be formulated foradministration via various miscellaneous routes, for example, topical(i.e., transdermal) administration, mucosal administration (intranasal,vaginal, etc.) and/or inhalation.

Pharmaceutical compositions for topical administration may include theactive compound formulated for a medicated application such as anointment, paste, cream or powder. Ointments include all oleaginous,adsorption, emulsion and water-solubly based compositions for topicalapplication, while creams and lotions are those compositions thatinclude an emulsion base only. Topically administered medications maycontain a penetration enhancer to facilitate adsorption of the activeingredients through the skin. Suitable penetration enhancers includeglycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones andluarocapram. Possible bases for compositions for topical applicationinclude polyethylene glycol, lanolin, cold cream and petrolatum as wellas any other suitable absorption, emulsion or water-soluble ointmentbase. Topical preparations may also include emulsifiers, gelling agents,and antimicrobial preservatives as necessary to preserve the activeingredient and provide for a homogenous mixture. Transdermaladministration of the present invention may also comprise the use of a“patch”. For example, the patch may supply one or more active substancesat a predetermined rate and in a continuous manner over a fixed periodof time.

In certain embodiments, the pharmaceutical compositions may be deliveredby eye drops, intranasal sprays, inhalation, and/or other aerosoldelivery vehicles. Methods for delivering compositions directly to thelungs via nasal aerosol sprays has been described e.g., in U.S. Pat.Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein byreference in its entirety). Likewise, the delivery of drugs usingintranasal microparticle resins (Takenaga et al., 1998) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,specifically incorporated herein by reference in its entirety) are alsowell-known in the pharmaceutical arts. Likewise, transmucosal drugdelivery in the form of a polytetrafluoroetheylene support matrix isdescribed in U.S. Pat. No. 5,780,045 (specifically incorporated hereinby reference in its entirety).

The term aerosol refers to a colloidal system of finely divided solid ofliquid particles dispersed in a liquefied or pressurized gas propellant.The typical aerosol of the present invention for inhalation will consistof a suspension of active ingredients in liquid propellant or a mixtureof liquid propellant and a suitable solvent. Suitable propellantsinclude hydrocarbons and hydrocarbon ethers. Suitable containers willvary according to the pressure requirements of the propellant.Administration of the aerosol will vary according to subject's age,weight and the severity and response of the symptoms.

VIII. DIAGNOSTICS

The antagonist or related-compound can be used for diagnosing,monitoring, or prognosis of spinal cord injury, for example monitoringthe damage to neurons, or in monitoring neuronal cells in zones ofedema, etc.

A. Genetic Diagnosis

One embodiment of the instant invention comprises a method for detectingexpression of any portion of a NC_(Ca-ATP) channel, for example,expression of the regulatory unit, SUR1, and/or expression of thepore-forming subunit. This may comprise determining the level of SUR1expressed and/or the level of the pore-forming subunit expressed. It isunderstood by the present invention that the up-regulation or increasedexpression of the NC_(Ca-ATP) channel relates to increased levels ofSUR1, which correlates to increased neuronal damage, such as edema.

Firstly, a biological sample is obtained from a subject. The biologicalsample may be tissue or fluid. In certain embodiments, the biologicalsample includes cells from the spinal cord and/or endothelial cells ormicrovessels associated with the spinal cord or spinal tissue.

Nucleic acids used are isolated from cells contained in the biologicalsample, according to standard methodologies (Sambrook et al., 1989). Thenucleic acid may be genomic DNA or fractionated or whole cell RNA. WhereRNA is used, it may be desired to convert the RNA to a complementary DNA(cDNA). In one embodiment, the RNA is whole cell RNA; in another, it ispoly-A RNA. Normally, the nucleic acid is amplified.

Depending on the format, the specific nucleic acid of interest isidentified in the sample directly using amplification or with a second,known nucleic acid following amplification. Next, the identified productis detected. In certain applications, the detection may be performed byvisual means (e.g., ethidium bromide staining of a gel). Alternatively,the detection may involve indirect identification of the product viachemiluminescence, radioactive scintigraphy of radiolabel or fluorescentlabel or even via a system using electrical or thermal impulse signals(Affymax Technology; Bellus, 1994).

Following detection, one may compare the results seen in a given subjectwith a statistically significant reference group of normal subjects andsubjects that have been diagnosed with a spinal cord injury and orsecondary injury associated therewith, etc.

Yet further, it is contemplated that chip-based DNA technologies such asthose described by Hacia et al., (1996) and Shoemaker et al., (1996) canbe used for diagnosis. Briefly, these techniques involve quantitativemethods for analyzing large numbers of genes rapidly and accurately. Bytagging genes with oligonucleotides or using fixed probe arrays, one canemploy chip technology to segregate target molecules as high densityarrays and screen these molecules on the basis of hybridization. Seealso Pease et al., (1994); Fodor et al., (1991).

B. Other Types of Diagnosis

In order to increase the efficacy of molecules, for example, compoundsand/or proteins and/or antibodies, as diagnostic agents, it isconventional to link or covalently bind or complex at least one desiredmolecule or moiety.

Certain examples of conjugates are those conjugates in which themolecule (for example, protein, antibody, and/or compound) is linked toa detectable label. “Detectable labels” are compounds and/or elementsthat can be detected due to their specific functional properties, and/orchemical characteristics, the use of which allows the antibody to whichthey are attached to be detected, and/or further quantified if desired.

Conjugates are generally preferred for use as diagnostic agents.Diagnostics generally fall within two classes, those for use in in vitrodiagnostics, such as in a variety of immunoassays, and/or those for usein vivo diagnostic protocols, generally known as “molecule-directedimaging”.

Many appropriate imaging agents are known in the art, as are methods fortheir attachment to molecules, for example, antibodies (see, for e.g.,U.S. Pat. Nos. 5,021,236; 4,938,948; and 4,472,509, each incorporatedherein by reference). The imaging moieties used can be paramagneticions; radioactive isotopes; fluorochromes; NMR-detectable substances;X-ray imaging.

In the case of paramagnetic ions, one might mention by way of exampleions such as chromium (III), manganese (II), iron (III), iron (II),cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and/or erbium (III), with gadoliniumbeing particularly preferred. Ions useful in other contexts, such asX-ray imaging, include but are not limited to lanthanum (III), gold(III), lead (II), and especially bismuth (III).

In the case of radioactive isotopes for therapeutic and/or diagnosticapplication, one might mention astatine²¹¹, ¹¹carbon, ¹⁴carbon,⁵¹chromium, ³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt, copper⁶⁷, ¹⁵²Eu, gallium⁶⁷,³hydrogen, iodine¹²³, iodine ¹²⁵, iodine¹³¹, indium¹¹¹, ⁵⁹iron,³²phosphorus, rhenium¹⁸⁶, rhenium¹⁸⁸, ⁷⁵selenium, ³⁵sulphur,technicium^(99m) and/or yttrium⁹⁰. ¹²⁵I is often being preferred for usein certain embodiments, and technicium^(99m) and/or indium¹¹¹ are alsooften preferred due to their low energy and suitability for long rangedetection.

Among the fluorescent labels contemplated for use as conjugates includeAlexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL,BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM,Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, RhodamineRed, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or TexasRed.

Other types of conjugates contemplated in the present invention arethose intended primarily for use in vitro, where the molecule is linkedto a secondary binding ligand and/or to an enzyme (an enzyme tag) thatwill generate a colored product upon contact with a chromogenicsubstrate. Examples of suitable enzymes include urease, alkalinephosphatase, (horseradish) hydrogen peroxidase or glucose oxidase.Preferred secondary binding ligands are biotin and/or avidin andstreptavidin compounds. The use of such labels is well known to those ofskill in the art and are described, for example, in U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and4,366,241; each incorporated herein by reference.

The steps of various other useful immunodetection methods have beendescribed in the scientific literature, such as, e.g., Nakamura et al.,(1987). Immunoassays, in their most simple and direct sense, are bindingassays. Certain preferred immunoassays are the various types ofradioimmunoassays (RIA) and immunobead capture assay.Immunohistochemical detection using tissue sections also is particularlyuseful. However, it will be readily appreciated that detection is notlimited to such techniques, and Western blotting, dot blotting, FACSanalyses, and the like also may be used in connection with the presentinvention.

Immunologically-based detection methods for use in conjunction withWestern blotting include enzymatically-, radiolabel-, orfluorescently-tagged secondary molecules/antibodies against the SUR1 orregulatory subunit of the NC_(Ca-ATP) channel are considered to be ofparticular use in this regard. U.S. patents concerning the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated hereinby reference. Of course, one may find additional advantages through theuse of a secondary binding ligand such as a second antibody or abiotin/avidin ligand binding arrangement, as is known in the art.

In addition to the above imaging techniques, one of skill in the art isalso aware that positron emission tomography, PET imaging or a PET scan,can also be used as a diagnostic examination. PET scans involve theacquisition of physiologic images based on the detection of radiationfrom the emission of positrons. Positrons are tiny particles emittedfrom a radioactive substance administered to the subject.

Thus, in certain embodiments of the present invention, the antagonist orrelated-compound thereof is enzymatically-, radiolabel-, orfluorescently-tagged, as described above and used to diagnose, monitor,and/or stage neuronal damage in the spinal cord and/or predict or stagesecondary damage associated with the spinal cord injury. For example,the labeled antagonist or related-compound thereof may be used todetermine or define the penumbra or the areas at risk for damage after aspinal cord injury.

IX. DIAGNOSTIC OR THERAPEUTIC KITS

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, it is envisioned that a compound that selectivelybinds to or identifies SUR1 may be comprised in a diagnostic kit. Suchcompounds can be referred to as an “SUR1 marker”, which may include, butare not limited to antibodies (monoclonal or polyclonal), SUR1oligonucleotides, SUR1 polypeptides, small molecule or combinationsthereof, antagonist, etc. It is envisioned that any of these SUR1markers may be linked to a radioactive substance and/or a fluorescentmarker and/or a enzymatic tag for quick determination. The kits may alsocomprise, in suitable container means a lipid, and/or an additionalagent, for example a radioactive or enzymatic or florescent marker.

The kits may comprise a suitably aliquoted SUR1 marker, lipid and/oradditional agent compositions of the present invention, whether labeledor unlabeled, as may be used to prepare a standard curve for a detectionassay. The components of the kits may be packaged either in aqueousmedia or in lyophilized form. The container means of the kits willgenerally include at least one vial, test tube, flask, bottle, syringeor other container means, into which a component may be placed, andpreferably, suitably aliquoted. When there is more than one component inthe kit, the kit also will generally contain a second, third or otheradditional container into which the additional components may beseparately placed. However, various combinations of components may becomprised in a vial. The kits of the present invention also willtypically include a means for containing the SUR1 marker, lipid,additional agent, and any other reagent containers in close confinementfor commercial sale. Such containers may include injection or blowmolded plastic containers into which the desired vials are retained.

Therapeutic kits of the present invention are kits comprising anantagonist or a related-compound thereof. Thus, the kit may comprise anSUR1 antagonist or related-compound thereof to block and/or inhibit theNC_(Ca-ATP) channel. Such kits will generally contain, in suitablecontainer means, a pharmaceutically acceptable formulation of SUR1antagonist or related-compound thereof. The kit may have a singlecontainer means, and/or it may have distinct container means for eachcompound.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. The SUR1 antagonist orrelated-compounds thereof may also be formulated into a syringeablecomposition. In which case, the container means may itself be a syringe,pipette, and/or other such like apparatus, from which the formulationmay be applied to an infected area of the body, injected into an animal,and/or even applied to and/or mixed with the other components of thekit.

Examples of aqueous solutions include, but are not limited to ethanol,DMSO and/or Ringer's solution. In certain embodiments, the concentrationof DMSO, polyethylene glycol (PEG) or ethanol that is used is no greaterthan 0.1% or (1 ml/1000 L).

However, the components of the kit may be provided as dried powder(s).When reagents and/or components are provided as a dry powder, the powdercan be reconstituted by the addition of a suitable solvent. It isenvisioned that the solvent may also be provided in another containermeans.

The container means will generally include at least one vial, test tube,flask, bottle, syringe and/or other container means, into which the SUR1antagonist or related-compounds thereof is suitably allocated. The kitsmay also comprise a second container means for containing a sterile,pharmaceutically acceptable buffer and/or other diluent.

The kits of the present invention will also typically include a meansfor containing the vials in close confinement for commercial sale, suchas, e.g., injection and/or blow-molded plastic containers into which thedesired vials are retained.

Irrespective of the number and/or type of containers, the kits of theinvention may also comprise, and/or be packaged with, an instrument forassisting with the injection/administration and/or placement of the SUR1antagonist or related-compounds thereof within the body of an animal.Such an instrument may be a syringe, pipette, forceps, and/or any suchmedically approved delivery vehicle.

In addition to the SUR1 antagonist or related-compounds thereof, thekits may also include a second active ingredient. Examples of the secondactive ingredient include substances to prevent hypoglycemia (e.g.,glucose, D5W, glucagon, etc.), and steroids (e.g., methylprednisolone),etc. These second active ingredients may be combined in the same vial asthe SUR1 antagonist or related-compounds thereof or they may becontained in a separate vial.

Still further, the kits of the present invention can also includeglucose testing kits. Thus, the blood glucose of the patient is measuredusing the glucose testing kit, then the SUR1 antagonist orrelated-compounds thereof can be administered to the subject followed bymeasuring the blood glucose of the patient.

In addition to the above kits, the therapeutic kits of the presentinvention can be assembled such that an IV bag comprises a septum orchamber which can be opened or broken to release the compound into theIV bag. Another type of kit may include a bolus kit in which the boluskit comprises a pre-loaded syringe or similar easy to use, rapidlyadministrable device. An infusion kit may comprise the vials or ampoulesand an IV solution (e.g., Ringer's solution) for the vials or ampoulesto be added prior to infusion. The infusion kit may also comprise abolus kit for a bolus/loading dose to be administered to the subjectprior, during or after the infusion.

X. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Modulation of NC_(Ca-ATP) Channel

When a cell is depolarized by a massive influx of Na⁺, H₂O is drawn intothe cell due to the osmotic gradient. Influx of H₂O causes cellblebbing, i.e., cytoxic edema. R1 astrocytes were examined for thisphenomenon using scanning electron microscopy (SEM) and phase contrastmicroscopy. Freshly isolated cells examined with SEM showed a complexsurface decorated with multiple fine processes (FIG. 1A). Shortly afterexposure to Na azide, but well after depolarization is expected, thecomplex cell surface began to be replaced by surface blebs accompaniedby smoothing out of the membrane (FIG. 1B). Later, the surfaceappearance was dominated by blebs, with complete loss of the delicateprocesses observed in controls (FIG. 1C).

Blebbing is reproduced in the absence of ATP depletion by simply openingthe NC_(Ca-ATP) channel with diazoxide (FIG. 2). Conversely, blebbingtypically observed with Na azide-induced ATP depletion is completelyprevented by glibenclamide (FIG. 2). Blebbing and cytotoxic edemapresage necrotic cell death.

Example 2 Modulation by Estrogen

A characteristic feature of K_(ATP) channels (Kir6.1, Kir6.2) is thatchannel affinity for ATP is modulated by the presence of the membranelipid, phosphatidylinositol 4,5-bisphosphate (PIP₂). The open-statestability of K_(ATP) channels is increased by application of PIP₂ to thecytoplasmic side of the membrane (Ashcroft, 1998; Baukrowitz et al.,1998; Rohacs et al., 1999). An increase in the open-state stability ismanifested as an increase in the channel open probability in the absenceof ATP, and in a corresponding decrease in sensitivity to inhibition byATP (Enkvetchakul et al., 2000; Haruna et al., 2000; Koster et al.,1999; and Larsson et al., 2000).

Given the numerous similarities between the K_(ATP) channel and theNC_(Ca-ATP) channel, the inventors postulated that ATP-sensitivity ofthe NC_(Ca-ATP) channel would respond to PIP₂ in the same way. This wastested by studying NC_(Ca-ATP) channels in inside out patches with Cs⁺as the charge carrier, and with 1 μM Ca²⁺ and 10 μM ATP in the bath,with the latter expected to fully block the channel. Under theseconditions, only the NC_(Ca-ATP) channel was recorded in R1 astrocytes.When PIP₂ (50 μM) was added to the bath, channel activity becameprominent (FIG. 3), as predicted by analogy to the effect of PIP₂ onK_(ATP) channels. This channel activity was blocked by glibenclamide,confirming identity of the channel.

To determine if a receptor-mediated mechanism was involved in themodulation of NC_(Ca-ATP) channel activity, a well known phospholipase C(PLC) was used to study if PLC activation would cause degradation andconsumption of PIP₂ and thereby increase affinity for ATP, e.g., reducechannel opening. Estrogen is a well known PLC activator in brain as wellas elsewhere (Beyer et al., 2002; Le Mellay et al., 1999; Qui et al.,2003). For this experiment, cell attached patches were studied toprevent alteration of intracellular signaling machinery. NC_(Ca-ATP)channel activity was produced by exposure to Na azide to cause depletionof cellular ATP (FIG. 4, initial part of the record).

When estrogen (E2; 10 nM) was applied to the bath, activity due to theNC_(CA-ATP) channel was soon terminated (FIG. 4). This suggested thatestrogen exerted regulatory control over the NC_(Ca-ATP) channel, andsuggested that an estrogen receptor capable of rapid (non-genomic)activation of signaling cascades was present on these cells.

Next, to determine whether estrogen receptors could be detected in R1astrocytes from males and females, gelatin sponge implants wereharvested 7 days after implantation in a group of 3 female rats (F) andanother group of 3 male rats (M). Pooled protein from each group wasanalyzed at 2 dilutions (4×=50 μg total protein; 1×=12.5 μg totalprotein) by Western blotting, with protein from uterus being used as acontrol (FIG. 5A). Membranes were blotted with an antibody thatrecognized both α and β estrogen receptors. Both males and femalesshowed prominent bands at the appropriate molecular weights for the α(66 kDa) and β (55 kDa) receptors (FIG. 5) (Hiroi et al., 1999). Thesame samples of protein from males and females were also used to confirmpresence of SUR1, with protein from pancreas used as a positive control(FIG. 5B). Notably, estrogen receptors have previously been reported inastrocytes from males and females (Choi et al., 2001). In cerebralcortex, the β form is reportedly more abundant (Guo et al., 2001) assuggested by the Western blot.

Next, the electrophysiological experiment of FIG. 4 was repeated usingR1 astrocytes harvested from male rats. As above, cell attached patcheswere studied in which NC_(Ca-ATP) channel activity was activated bydepletion of intracellular ATP following exposure to Na azide (FIG. 6A).Examination of the record at higher temporal resolution confirmedactivity of a well defined channel of the appropriate conductance forthe NC_(Ca-ATP) channel (FIG. 6B). When estrogen was applied to the bath(FIG. 6, E2, 10 nM, arrow), activity due to the NC_(Ca-ATP) channel wasquickly terminated (FIG. 6). These data provided further evidence thatestrogen exerted regulatory control over the NC_(Ca-ATP) channel, andsuggested, in addition, that this response was equally robust in R1astrocytes from males and females.

By analogy to the effects of estrogen, other mechanisms that depletePIP₂, including other receptor-mediated mechanism as well as more directactivators of PLC such as G-proteins etc., would be expected to have asimilar inhibitory effect on activity of the NC_(Ca-ATP) channel andthereby exert a protective effect.

Example 3 NC_(Ca-ATP) Channel and Necrotic Death

Applicants discovered a new mechanism of necrotic death of reactiveastrocytes in brain injury and stroke that implicate an important rolein spinal cord injury. Blebbing and cytotoxic edema presage necroticcell death. Freshly isolated reactive astrocytes were labeled withpropidium iodide, a marker of necrotic death, and for annexin V, amarker of apoptotic death. Cells exposed to Na azide showed a markedincrease in necrotic but not apoptotic death (FIG. 7). However, whenglibenclamide was present, Na azide-induced necrotic cell death wassignificantly reduced (FIG. 7). These in vitro data show the importantrole of the NC_(Ca-ATP) channel in necrotic death of reactiveastrocytes, and indicate antagonists of SUR1, such as glibenclamide, areuseful in preventing cytotoxic edema and necrotic death in vivo.

The Applicants studied NC_(Ca-ATP) channels in a rodent model of stroke.In the penumbra, SUR1 labeling was found in stellate-shaped cells (FIG.8A) that were also GFAP-positive. In the middle of the stroke, stellatecells were absent, but SUR1 labeling was found in round cells exhibitinga bleb-like appearance (FIG. 8B,C) that were also GFAP-positive. Theround cells with blebbing in situ resembled reactive astrocytes in vitroundergoing necrotic death after exposure to Na azide. The effect ofglibenclamide vs. saline was examined, administered viasubcutaneously-implanted osmotic mini-pump (300 μM at 0.5 μl/hr). Insaline treated rats, 3-day mortality after stroke was 68%, whereas inglibenclamide-treated rats, 3-day mortality was reduced to 28% (n=20 ineach group; p<0.001, by χ²). In separate animals, it was found that thestroke hemisphere in glibenclamide-treated rats contained only half asmuch excess water as in saline-treated rats (n=5 in each group; p<0.01,by t-test), confirming an important role of the NC_(Ca-ATP) channel inedema formation.

The Applicants also studied SUR1 in a rodent model of trauma. The effectof direct infusion of drugs into the site of trauma using an implantedosmotic mini-pump was examined. The channel inhibitor, glibenclamide,was used to reduce death of reactive astrocytes, and the channelactivator, diazoxide, to promote astrocyte death. Briefly, it was foundthat glibenclamide infusion reduced the overall injury response,stabilized the gliotic capsule around the foreign body implant, andminimized the inflammatory response compared to control.

Conversely, diazoxide essentially destroyed the gliotic capsule andincited a huge inflammatory response, characterized by massive influx ofPMNs (FIG. 9A, B). These data suggested that NC_(Ca-ATP) channel plays acritical role in the injury response, and they strongly supported thehypothesis that inflammation was closely linked to activity of theNC_(CA-ATP) channel and necrotic death of reactive astrocytes.

Example 4 Expression of Functional NC_(CA-ATP) Channels in Spinal CordContusion

SUR1 in a rodent model of spinal cord contusion was identified.Immunolabeled spinal cord sections showed a large increase in SUR1expression in the region of injury (FIG. 10B), compared to control (FIG.10A). SUR1 was co-localized with GFAP (FIG. 10C), confirming involvementof reactive astrocytes. Examination of cells at high power confirmedthat SUR1-positive cells were stellate-shaped (FIG. 10D) GFAP-positivecells, consistent with the hypothesis that reactive astrocytes in spinalcord injury express the NC_(Ca-ATP) channel.

Further characterization of reactive astrocytes is carried out byisolating reactive astrocytes from contused spinal cord 3-5 days afterinjury using fluorescence-assisted cell sorting (FACS). Freshly isolatedcells are patch clamped to demonstrate channels with expectedphysiological and pharmacological properties. A spinal cord injury (SCI)model is used and includes use of the NYU-style impactor (Yu et al.,2001). Reactive astrocytes are isolated from enzymatically dispersedspinal cord tissue using anti-SUR1 antibody and FACS. Use of FACS forisolation of another subtype of reactive astrocyte in brain injury(Dalton et al., 2003). Patch clamp methods are used to measure singlechannel conductance, sensitivity to ATP, and sensitivity toglibenclamide and diazoxide, as described astrocytes isolated from braininjury (Chen et al., 2001; Chen and Simard, 2003).

Example 5 Block of SUR1 Prevents Delayed Hemorrhagic Conversion

The lesion in spinal cord contusion results not only from physicaltrauma to the tissues, but also from secondary damage that causesexpansion of the original lesion and worsens neurological compromise.Mechanisms of secondary are generally attributed to development ofischemia, edema, release of excitatory amino acids, oxidative injury andinflammation. The Applicants discovered that hemorrhage also is a keycomponent of this process of secondary injury. Hemorrhage expands afterinjury, because of progressive pathological involvement of capillaries,a phenomenon that is termed “hemorrhagic conversion”.

To study the role of the SUR1-regulated NC_(CA-ATP) channel in SCI, ahemi-cervical spinal cord contusion model was used. For this model, a 10gm weight is dropped 2.5 cm onto the left half of the exposed dura atC4-5 in adult female Long-Evans rats. Histopathological study 24 hrafter injury showed abundant up-regulation of SUR1 in capillariessurrounding the area of injury that was not present in controls (FIG.11). In addition, capillaries in the injury site that showedup-regulation of SUR1 were also found to express vimentin (FIG. 12), anintermediate filament protein commonly associated with astrocytes, butthat is also expressed by injured capillary endothelial cells in brainand spinal cord.

To provide further molecular evidence for involvement of SUR1, tissuesin spinal cord injury were also examined for the transcription factor,SP1, which is the principal transcription factor known to regulateexpression of SUR1. Immunolabeling of tissues in the region of injuryshowed prominent up-regulation of SP1 (FIG. 13B), compared to control(FIG. 13A).

To assess the role of newly expressed SUR1 in SCI, 2 groups of animalswere studied, one control- and one treatment-group, both of whichunderwent hemicervical spinal cord contusion plus post-injuryimplantation (blinded) of a miniosmotic pump that deliveredsubcutaneously either saline or the selective SUR1 blocker, low-doseglibenclamide (300 μM solution delivered at 0.5 μl/hr s.q.). Study 24 hrafter injury showed that, compared to controls, glibenclamide-treatedanimals had significantly less blood in the contusion site (FIG. 14A,B).Also, homogenates of spinal cord tissue showed significantly lesscoloration from hemoglobin/hemosiderin (FIG. 14C). Quantitative study ofhemoglobin concentration as a function of time after spinal cordcontusion showed a progressive increase over the first 6 hours afterinjury in saline-treated animals that was significantly ameliorated bytreatment with glibenclamide (FIG. 15)

Next, the lesions in the two groups of animals were assessed using GFAPto label reactive astrocytes and eriochrome cyanine-R to label myelin.Study 24 hr after injury showed that, compared to controls,glibenclamide-treated animals had significantly smaller lesions,significantly reduced GFAP expression, and significantly betterpreservation of contralateral long tracts (FIG. 16).

Yet further, behavioral assessments in the two groups of animals wereperformed. The animals were video-taped and vertical exploratorybehavior was quantified in an environment that animals had not beenpreviously exposed to. Study 24 hr after injury showed that, compared tocontrols, glibenclamide-treated animals exhibited significantly improvedvertical exploratory behavior (FIG. 17).

Recognition of the phenomenon of delayed hemorrhagic conversion inspinal cord contusion provides an extraordinary opportunity to reducesecondary damage. As is widely known, blood is severely toxic to CNStissues, and is responsible for formation of edema, generation ofreactive oxidative species, and inciting inflammation. The concept ofdelayed hemorrhagic conversion is a novel concept in SCI, and discoverythat glibenclamide can be used to ameliorate this condition provides anunprecedented opportunity to improve outcome by reducing secondarydamage.

Example 6 Role of NC_(Ca-ATP) Channels in Cytotoxic Edema and NecroticDeath of Astrocytes and Release of Biologically Active Molecules thatPromote Tissue Inflammation

Identification of candidate intracellular molecule(s) released by cellmembrane lysis during necrotic cell death that play a role in initiatingan inflammatory response in spinal cord injury is also contemplated inthe present invention. Reactive astrocytes are isolated from contusedspinal cord 3-5 days after injury using FACS. Using freshly isolatedcells, the effect of Na azide (1 mM) vs. Na azide plus glibenclamide (1μM) on necrotic vs. apoptotic cell death during the first 3 hr afterpoisoning is assessed by: performing morphological studies using phasecontrast microscopy, scanning electron microscopy and transmissionelectron microscopy; labeling for propidium iodide vs. annexin V; andassessing DNA degradation using TUNEL labeling and DNA laddering.

Using freshly isolated cells, ELISAs are used to measure release ofHSP-32 and HSP-70 following necrotic death of astrocytes induced by Naazide. Also, using the same experimental paradigm, we will assess theprotective effect of glibenclamide on Na azide-induced release of HSP-32and HSP-70. Standard FACS methods are used as well as scanning andtransmission electron microscopy and phase contrast microscopy, whichallows an individual cell to be followed sequentially during blebbing.Immunofluorescence is also used, as described herein.

Example 7 Ability of Antagonist of NC_(Ca-ATP) Channel to ReduceInflammatory Response in Spinal Cord Contusion In Vivo

Tissues are studied about 3 days after injury. In rats with spinal cordcontusion treated with either saline or glibenclamide, the inflammatoryresponse in situ is assessed using qualitative immunofluorescencelabeling for activated microglia (OX-42), macrophages (MAC-387, Novus),PMNs (MMP-8, Chemicon) and iNOS. For these experiments, fresh-frozensections of spinal cord adjacent to and in the area of contusion arestudied. Quantitative FACS analysis for macrophages (MAC-387) and PMNs(MMP-8) are performed. For these experiments, a 15-mm segment of spinalcord containing the area of contusion is obtained and enzymaticallydispersed for FACS analysis; quantitative Western blots for SUR1 and foriNOS. For these experiments, a 15-mm segment of spinal cord containingthe area of contusion is obtained and homogenized for Western blotting.Standard methods and materials are used, as described above, includingFACS analysis, Western blots and immunofluorescence imaging.

REFERENCES

All patents and publications mentioned in the specifications areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

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Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

1. A method of treating a subject suffering from a spinal cord injurycomprising administering to the subject a compound effective to inhibita NC_(Ca-ATP) channel in a neuronal cell, a neuroglia cell, a neuralendothelial cell or a combination thereof.
 2. The method of claim 1,wherein the compound reduces cell death.
 3. The method of claim 1,wherein the compound reduces the inflammatory response.
 4. The method ofclaim 1, wherein the compound reduces hemorrhagic conversion.
 5. Themethod of claim 1, wherein the compound reduces secondary damageassociated with the spinal cord injury.
 6. The method of claim 1,wherein the compound is a type 1 sulfonylurea receptor (SUR1)antagonist.
 7. The method of claim 6, wherein the SUR1 antagonist isselected from the group consisting of glibenclamide, tolbutamide,repaglinide, nateglinide, meglitinide, midaglizole, LY397364, LY389382,glyclazide, glimepiride, estrogen, and estrogen related-compounds. 8.The method of claim 6, wherein the amount of SUR1 antagonistadministered to the subject is in the range of about 0.0001 μg/kg/day toabout 20 mg/kg/day.
 9. The method of claim 6, wherein the amount of SUR1antagonist administered to the subject is in the range of about 0.01g/kg/day to about 100 μg/kg/day.
 10. The method of claim 6, wherein theamount of SUR1 antagonist administered to the subject is in the range ofabout 100 μg/kg/day to about 20 mg/kg/day.
 11. The method of claim 6,wherein the SUR1 antagonist is administered as a bolus injection. 12.The method of claim 6, wherein the SUR1 antagonist is administered as aninfusion.
 13. The method of claim 6, wherein the SUR1 antagonist isadministered as a bolus injection in combination with an infusion. 14.The method of claim 6, wherein the amount of SUR1 antagonistadministered to the subject is in the range of about 0.000μg/kg/treatment to about 20 mg/kg/treatment.
 15. The method of claim 6,wherein the amount of SUR1 antagonist administered to the subject is inthe range of about 0.01 μg/kg/treatment to about 100 μg/kg/treatment.16. The method of claim 6, wherein the amount of SUR1 antagonistadministered to the subject is in the range of about 100 μg/kg/treatmentto about 20 mg/kg/treatment.
 17. The method of claim 6, wherein the SUR1antagonist blocks the influx of Na+ into the cells thereby preventingdepolarization of the cells.
 18. The method of claim 6, wherein the SUR1antagonist blocks the influx of Na+ into the cells thereby preventingcytotoxic edema.
 19. The method of claim 1, wherein the compound isadministered alimentary or parenterally.
 20. The method of claim 19,wherein alimentary comprises orally, buccally, rectally or sublingually.21. The method of claim 19, wherein parenterally comprisesintravenously, intradermally, intramuscularly, intraarterially,intrathecally, subcutaneously, intraperitoneally, or intraventricularly.22. The method of claim 1, wherein the compound is administeredmucosally.
 23. The method of claim 22, wherein mucosally comprisesintranasally.
 24. The method of claim 1, wherein inhibition of theNC_(Ca-ATP) channel results in a decrease in the morbidity of thesubject.
 25. The method of claim 1, wherein inhibition of theNC_(Ca-ATP) channel results in a decrease in extravasated blood near thecontusion site in the subject.
 26. The method of claim 1, whereininhibition of the NC_(Ca-ATP) channel reduces the size of the lesion onthe spinal cord.
 27. The method of claim 24, wherein the reduction inthe size of the lesion reduces contralateral involvement of the spinalcord.
 28. The method of claim 1, wherein inhibition of the NC_(Ca-ATP)channel decreases the up-regulation of GFAP.
 29. The method of claim 1,wherein inhibition of the NC_(Ca-ATP) channel in a neuronal cell, aneuroglia cell, an endothelial cell or a combination thereof preservesmyelinated long tracts.
 30. The method of claim 1, wherein inhibition ofthe NC_(Ca-ATP) channel improves the movement or sensation by thesubject.
 31. A method of reducing edema in the penumbra of the spinalcord injury in a subject comprising administering to the subject acompound effective to inhibit a NC_(Ca-ATP) channel in a neuronal cell,a neuroglia cell, a neural endothelial cell or a combination thereof.32. A method of treating a subject at risk for a developing a spinalcord injury comprising administering to the subject a compound effectiveto inhibit a NC_(Ca-ATP) channel in a neuronal cell, a neuroglia cell, aneural endothelial cell or a combination thereof.
 33. The method ofclaim 32, wherein the subject is undergoing a surgical treatment. 34.The method of claim 32, wherein the subject is undergoing radiationtreatment.
 35. A method of reducing extravasation of blood from a spinalcord injury comprising administering to the subject a compound effectiveto inhibit a NC_(Ca-ATP) channel in a neuronal cell, a neuroglia cell, aneural endothelial cell or a combination thereof.
 36. The method ofclaim 35, wherein the compound is a type 1 sulfonylurea receptor (SUR1)antagonist.
 37. The method of claim 36, wherein the SUR1 antagonist isselected from the group consisting of glibenclamide, tolbutamide,repaglinide, nateglinide, meglitinide, midaglizole, LY397364, LY389382,glyclazide, glimepiride, estrogen, and estrogen related-compounds. 38.The method of claim 35, wherein the subject is at risk for a spinal cordinjury.
 39. The method of claim 38, wherein the compound is administeredbefore, during or after a surgical or radiation treatment.
 40. A methodof diagnosing neuronal cell edema and/or cytotoxic damage in the spinalcord of a subject comprising: labeling an antagonist of SUR1;administering the labeled antagonist of SUR1 to the subject; measuringthe levels of labeled antagonist of SUR1 in the spinal cord of thesubject, wherein the presence of labeled antagonist of SUR1 in thespinal cord of the subject indicates neuronal cell edema and/orcytotoxic damage in the spinal cord.
 41. A method of determining thepenumbra following spinal cord injury in a subject comprising: labelingan antagonist of SUR1; administering the labeled antagonist of SUR1 tothe subject; visualizing the labeled antagonist of SUR1 in the spinalcord of the subject, wherein the presence of labeled antagonist of SUR1indicates the penumbra following a spinal cord injury in the subject.42. The method of claim 41, wherein determining the penumbra indicatesthe position of neuronal damage.
 43. The method of claim 41, whereindetermining the penumbra monitors disease progression.